foWn 


TEE-PRINCIPLES 


OF 


BACTERIOLOQYr 

A  PRACTICAL  MANUAL  FOR  STUDENTS 
AND  PHYSICIANS. 


BY 


A.  C.  ABBOTT,  M.  D., 

PROFESSOR  OF  HYGIENE  AND  BACTERIOLOGY,    AND  DIRECTOR   OF  THE  LABO- 
RATORY  OF   HYGIENE,   UNIVERSITY   OF  PENNSYLVANIA. 


SEVENTH  EDITION, 

ENLARGED    AND    THOROUGHLY    REVISED. 
With  100  Illustrations,  of  which  24  are  colored. 


LEA  BROTHERS  &  CO., 
PHILADELPHIA    AND    NEW    YORK, 
1905, 


A  & 
\  q  o5 

01OLOGY 


Enteredaccording  to  Act  of  Congress  in  the  year  1905,  by 

LEA   BROTHERS   &   CO., 
in  the  Office  of  the  Librarian  of  Congress.    All  rights  reserved. 


WESTCOTT    &    THOMSON, 
ELECTROTYPERS,    PHILADA. 


PRESS  OF 
W.   J.   DORNAN,   PHILADA. 


PREFACE  TO  SEVENTH  EDITION. 


DURING  the  interval  that  has  elapsed  since  the  appear- 
ance of  the  Sixth  Edition  of  this  book  the  problem  that 
has  chiefly  engaged  the  attention  of  the  foremost  workers 
in  the  field  of  Bacteriology  is  the  old  one  of  Infection 
and  Immunity. 

In  its  modern  development  this  has  become  a  question 
of  the  broadest  biological  bearing,  and,  as  knowledge 
accumulates,  we  find  it  to  be  no  longer  of  >  exclusive 
bacteriological  interest. 

Ramifying,  as  it  does,  in  many  diverse  fields  it  grows 
correspondingly  complex,  and  a  complete  presentation 
of  the  conceptions  and  methods  would  be  inappropriate 
to  a  work  of  this  character.  However,  an  effort  has 
been  made  to  present  in  this  edition  sufficient  to  acquaint 
the  reader  with  the  manifold  directions  taken  by  these 
studies. 

Furthermore,  the  practical  results,  often  of  the  utmost 
importance  to  Preventive  Medicine,  have  been  included. 
This,  we  believe,  adds  materially  to  the  value  of  the  book. 

The  section  on  Methods  has  been  brought  fully  up  to 
date,  and,  while  not  covering  all  the  new  technique, 

iii 

144174 


iv  PREFACE  TO  SEVENTH  EDITION. 

contains  those  procedures  which  have  been  tried  and  found 
trust  worthy. 

In  the  descriptive  section  certain  less  important  species 
and  varieties  have  been  eliminated,  and  those  of  more 
frequent  occurrence  substituted. 

Nomenclature  has  been  altered  to  conform  to  the 
suggestions  of  the  advanced  systematists. 

In  presenting  this  edition  it  gives  me  great  pleasure 
to  acknowledge  the  valuable  assistance  rendered  by 
Prof.  D.  H.  Bergey,  of  this  Laboratory. 

A.  C.  A. 

LABORATORY  OF  HYGIENE, 
UNIVERSITY  OF  PENNSYLVANIA. 


PREFACE  TO  THE  FIRST  EDITION. 


IN  preparing  this  book  the  author  has  kept  in  mind 
the  needs  of  the  student  and  practitioner  of  medicine, 
for  whom  the  importance  of  an  acquaintance  with  prac- 
tical bacteriology  cannot  be  overestimated. 

It  is  to  advances  made  through  bacteriological  re- 
search that  we  are  indebted  for  much  of  our  knowledge 
of  the  conditions  underlying  infection,  and  for  the  elu- 
cidation of  many  hitherto  obscure  problems  concerning 
the  etiology,  the  modes  of  transmission,  and  the  means 
of  prevention  of  infectious  maladies. 

Only  within  a  comparatively  short  time  have  students 
and  physicians  been  enabled  to  obtain  the  systematic 
instruction  in  this  science  that  is  of  value  in  aiding 
them  in  their  efforts  to  check  disease.  The  rapid  in- 
crease in  the  number  who  are  availing  themselves  of 
these  opportunities  speaks  directly  for  the  practical 
value  of  the  science. 

As  the  majority  of  those  undertaking  the  study  of 
bacteriology  do  so  with  the  view  of  utilizing  it  in  med- 
ical practice,  and  as  many  of  these  can  devote  to  it  but 
a  portion  of  their  time,  it  is  desirable  that  the  subject- 
matter  be  presented  in  as  direct  a  manner  as  possible. 

v 


vi  PREFACE  TO  THE  FIRST  EDITION. 

Presuming  the  reader  to  be  unfamiliar  with  the  sub- 
ject, the  author  has  restricted  himself  to  those  funda- 
mental features  that  are  essential  to  its  understanding. 
The  object  has  been  to  present  the  important  ideas  and 
methods  as  concisely  as  is  compatible  with  clearness, 
and  at  the  same  time  to  accentuate  throughout  the 
underlying  principles  which  govern  the  work. 

With  the  view  of  inducing  independent  thought  on 
the  part  of  the  student,  and  of  diminishing  the  fre- 
quency of  that  oft-heard  query,  "  What  shall  I  do 
next?"  experiments  have  been  suggested  wherever  it 
is  possible.  These  have  been  arranged  to  illustrate  the 
salient  points  of  the  work  and  to  attract  attention  to 
the  minute  details,  upon  the  observation  of  which  so 
much  in  bacteriology  depends. 

PHILADELPHIA,  December.  1891. 


CONTENTS. 


INTRODUCTION. 

PAGES 

"Omne  vivum  ex  vivo" — The  overthrow  of  the  doctrine  of 
spontaneous  generation — Earlier  bacteriological  studies — 
The  birth  of  modern  bacteriology 17-30 

CHAPTER  I. 

Definition  of  bacteria— Difference  between  parasites  and 
saprophytes — Their  place  in  nature — Bacterial  enzymes — 
Products  of  bacteria — Nutrition  of  bacteria — Their  relation 
to  oxygen — Influence  of  temperature  upon  their  growth — 
Cheinotaxis 31-48 

CHAPTER  II. 

Morphology  of  bacteria — Chemical  composition  of  bacteria — 
Classification  of  bacteria  into  families  and  genera — Group- 
ing— Mode  of  multiplication — Spore-formation — Motility — 
The  thermal  death-point  of  bacteria 49-68 

CHAPTER  III. 

Principles  of  sterilization  by  heat — Methods  employed — Dis- 
continued sterilization — Fractional  sterilization — Appa- 
ratus employed — Sterilization  by  hot  air — Sterilization 
under  pressure — Chemical  disinfection  and  sterilization — 
Mode  of  action  of  disinfectants — Practical  disinfection  .  .  69-90 

CHAPTER  IV. 

Principles  involved  in  the  methods  of  isolation  of  bacteria 
in  pure  culture  by  the  plate  method  of  Koch — Materials 
employed 97-103 

CHAPTER  V. 

Preparation  of  nutrient  media — Bouillon,  gelatin,  agar-agar, 

potato,  blood-serum,  etc 104-137 


viii  CONTENTS. 

CHAPTER  VI. 

PAGES 

Preparation  of  the  tubes,  flasks,  etc.,  iu  which  the  media  are 

to  be  preserved 138-141 

CHAPTER  VII. 
Technique  of  making  plates— Petri  plates,  Esmarch  tubes,  etc.  142-151 

CHAPTER  VIII. 

The  incubating-oven— Gas-pressure  regulator — Thermo-regu- 

lator — Safety  burner  employed  in  heating  the  incubator    .    152-159 

CHAPTER  IX. 

The  study  of  colonies — Their  naked-eye  peculiarities  and 
their  appearance  under  different  conditions— Differences 
in  the  structure  of  colonies  from  different  species  of  bac- 
teria—Stab-cultures— Slant-cultures  160-166 

CHAPTER  X. 

Methods  of  staining — Impression  cover-slip  preparations — 
Solutions  employed — Preparation  and  staining  of  cover- 
slips — Staining  solutions — Staining  in  general — Special 
staining-methods 107-192 

CHAPTER  XI. 

Systematic  study  of  an  organism — Points  to  be  considered  in 
determining  the  morphologic  and  biologic  characters  of  a 
culture — Methods  by  which  the  various  biologic  and  chem- 
ical characters  of  a  culture  may  be  ascertained— Facts 
necessary  to  permit  the  identification  of  an  organism  as  a 
definite  species 193-234 

CHAPTER  XII. 

Inoculation  of  animals — Subcutaneous  inoculation — Intra- 
venous injection — Inoculation  in  to  the  great  serous  cavities 
and  into  the  anterior  chamber  of  the  eye — Observation  of 
animals  after  inoculation 235-257 

CHAPTER  XIII. 

Post-mortem  examination  of  animals — Bacteriological  exami- 
nation of  the  tissues — Disposal  of  tissues  and  disinfection 
of  instruments  after  the  examination — Study  of  tissues  and 
exndates  during  life 258-266 


CONTENTS.  ix 

APPLICATION  OF  THE  METHODS  OF 
BACTERIOLOGY.     DESCRIPTIONS 
OF  SOME  OF  THE  MORE  IM- 
PORTANT SPECIES. 

CHAPTER  XIV. 

PAGES 

To  obtain  material  with  which  to  begin  work 267-270 

CHAPTER  XV. 

The  pyogenic  organisms — Suppuration — Mirococcus  atireus — 
Micrococcus  pyogenes  and  citreus — Staphylococcus  epidermi- 
dis  albus — Streptococcus  pyogenes — Micrococcus  gonorrhcese— 
Micrococcus  intracellularis — Pseudomonas  a'eruginosa — Bacil- 
lus of  bubonic  plague — Bacterium  pseudodiphtheriticum  .  .  271-326 

CHAPTER  XVI. 

Sputum  septicaemia — Septicaemia  resulting  from  the  presence 
of  sarcina  tetrageua,  or  bacterium  pneumonias  in  the  spu- 
tum of  apparently  healthy  persons — The  occurrence  of 
bacterium  influenzas  in  sputum 327-345 

CHAPTER  XVII. 

Tuberculosis — Microscopic  appearance  of  miliary  tubercles — 
Diffuse  caseation— Cavity  formation— Encapsulation  of 
tuberculous  foci — Primary  infection — Modes  of  infection — 
Location  of  the  bacilli  in  the  tissues— Staining-peculiarities 
—Organisms  with  which  bacterium  tuberculosis  may  be  con- 
founded :  Bacterium  leprx  :  bacterium  smegmatis — Points  of 
differentiation — Acid-proof  bacteria — Actinomycetes — Acti- 
nomyces bovis,  Actinomyces  Israeli,  Actinomyces  madurse, 
Actinomyces  farcinicus,  Actinomyces  Eppingeri — Actinomyces 
pseudotuberculosis 346-388 

CHAPTER  XVIII. 

Glanders— Characteristics  of  the  disease— Histological  struc- 
ture of  the  glanders  nodule — Susceptibility  of  different 
animals  to  glanders — The  bacterium  of  glanders;  its  mor- 
phological and  cultural  peculiarities — Diagnosis  of  glanders  .  389-398 


CONTENTS. 


CHAPTER  XIX. 


Bacterium  diphtheriss—Its  isolation  and  cultivation — Mor- 
phological and  cultural  peculiarities — Pathogenic  properties 
— Variations  in  virulence — Bacterium  pseudodiphtheriti- 
cum — Bacterium  xerosis — Diphtheria  antitoxin 


399-4-25 


CHAPTER  XX. 

Typhoid  fever — Study  of  the  organism  concerned  in  its  pro- 
duction— Bacillus  coli — Its  resemblance  to  the  bacillus  of 
typhoid  fever — Its  morphological,  cultural,  and  pathogenic 
properties — Its  diffentiation  from  bacillus typhosus — Methods 
of  isolating  the  typhoid  bacillus— Bacillus  paratyphosis — Its 
resemblance  to  bacillus  typhosus — Its  morphological,  cul- 
tural, and  pathogenic  properties 

CHAPTER  XXI. 


426-463 


Bacillus  dysenteries — The  group  of  bacilli  found  in  cases  of 
epidemic,  endemic,  and  sporadic  dysentery — The  morpho- 
logical, biological,  and  pathogenic  characters  of  the  several 
members  of  the  group — The  differentiation  of  the  different 
types  of  bacilli 464-472 

CHAPTER  XXII. 

The  Cholera  Group  of  Organisms — The  spirillum  (comma 
bacillus)  of  Asiatic  cholera — Its  morphological  and  cultural 
peculari ties— Pathogenic  properties— The  bacteriological 
diagnosis  of  Asiatic  cholera — Microspira  Metchnikovi — 
Microspira  ("  vibrio")  Schuylkilliensis — Its  morphological, 


cultural,  and  pathogenic  characters 


473-509 


CHAPTER  XXIII. 

Study  of  bacterium  anthracis,  and  the  effects  produced  by  its 
inoculation  into  animals — Peculiarities  of  the  organism 
under  varying  conditions  of  surroundings — Anthrax  vac- 
cines— Anthrax  immune  serum 510-527 

CHAPTER  XXIV. 

The  most  important  of  the  organisms  found  in  the  soil — The 
nitrifying  bacteria— The  bacillus  of  tetanus— The  bacillus 
of  malignant  oedema— The  bacillus  of  symptomatic  anthrax 
—Bacterium  Welchii — Bacillus  sporogenes 528-558 


CONTENTS.  xi 

CHAPTER  XXV. 

PAGES 

Infection  and  immunity — The  types  of  infection ;  intimate 
nature  of  infection — Septicaemia,  toxaemia,  variations  in  in- 
fectious processes — Immunity,  natural  and  acquired,  active 
and  passive — The  hypotheses  that  have  been  advanced  in 
explanation  of  immunity — Conclusions 559-615 

CHAPTER  XXVI. 

Bacteriological  study  of  water — Methods  employed — Pre- 
cautions to  be  observed — Apparatus  used,  and  methods 
of  using  it — Methods  of  investigating  air  and  soil — Bac 
teriological  study  of  milk — Methods  employed 616-648 

CHAPTER  XXVII. 

Various  experiments  in  sterilization  by  steam  and  by  hot  air  .    649-653 

CHAPTER  XXVIII. 

Methods  of  testing  disinfectants  and  antiseptics — Experiments 
illustrating  the  precautions  to  be  taken— Experiments  in 
skin-disinfection 654-667 


APPENDIX. 
Apparatus  necessary  in  a  beginner's  bacteriological  laboratory .  669-674 


\*gALIF»Sa£^ 

BACTERIOLOGY. 


INTBODUCTION. 

"  Omne  vivum  ex  vivo  " — The  overthrow  of  the  doctrine  of  spontaneous 
generation — Earlier  bacteriological  studies — The  birth  of  modern 
bacteriology. 

BACTERIOLOGY  may  be  said  to  have  had  its  begin- 
ning with  the  observations  of  Leeuwenhoek  in  the 
latter  part  of  the  seventeenth  century.  Though  its 
most  rapid  and  important  development  has  taken  place 
since  about  1880,  still,  a  review  of  the  various  evo- 
lutionary phases  through  which  it  has  passed  in  the 
course  of  more  than  two  hundred  years  reveals  an 
entertaining  and  instructive  history.  From  the  very 
outset  its  history  is  inseparably  connected  with  that 
of  medicine,  and  from  the  outcome  of  bacteriological 
research  preventive  medicine,  in  its  modern  concep- 
tion, received  its  primary  impulse.  Through  a  more 
intimate  acquaintance  with  the  biological  activities 
of  the  unicellular  vegetable  micro-organisms  modern 
hygiene  has  attained  almost  the  dignity  of  an  exact 
science,  and  properly  merits  the  importance  and  promi- 
nence now  generally  accorded  to  it.  From  studies  in 
the  domain  of  bacteriology  our  knowledge  of  the  causa- 
tion, course,  and  prevention  of  infectious  diseases  is 
daily  becoming  more  accurate,  and  it  is  needless  to  em- 
phasize the  relation  of  such  knowledge  to  the  manifold 
problems  that  present  themselves  to  the  student  of 
2  17 


t    : BACTERIOLOGY. 

modern  med  cine.  Though  the  contributions  which 
have  done  most  to  place  bacteriology  on  the  footing  of 
a  science  are  those  of  recent  years,  still,  during  the 
earlier  stages  of  its  development,  many  observations 
were  made  which  formed  the  foundation-work  for  much 
that  was  to  follow.  Before  regularly  beginning  our 
studies,  therefore,  it  may  be  of  advantage  to  acquaint  our- 
selves with  the  more  prominent  of  those  investigations. 

Antony  van  Leeuwenhoek,  the  first  to  describe  the 
bodies  now  recognized  as  bacteria,  was  born  at  Delft,  in 
Holland,  in  1632.  He  was  not  considered  a  man  of 
liberal  education,  having  been  during  his  early  years  an 
apprentice  to  a  linendraper.  During  his  apprenticeship 
he  learned  the  art  of  lens-grinding,  in  which  he  became 
so  proficient  that  he  eventually  perfected  a  simple  lens 
by  means  of  which  he  was  enabled  to  see  objects  ^pf 
much  smaller  dimensions  than  any  hitherto  seen  $ith 
the  best  compound  microscopes  in  existence  at  that 
date.  At  the  time  of  his  discoveries  he  was  following 
the  trade  of  linendraper  in  Amsterdam. 

In  1675  he  published  the  fact  that  he  had  succeeded 
in  perfecting  a  lens  by  means  of  which  he  could  detect 
in  a  drop  of  rain-water  living,  motile  "  animalcules " 
of  the  most  minute  dimensions — smaller  than  anything 
that  had  hitherto  been  seen.  Encouraged  by  this  dis- 
covery, he  continued  to  examine  various  substances  for 
the  presence  of  what  he  considered  animal  life  in  its 
most  minute  form.  He  found  in  sea-water,  in  well- 
water,  in  the  intestinal  canal  of  frogs  and  birds,  and  in 
his  own  diarrhoeal  evacuations,  objects  that  differenti- 
ated themselves  the  one  from  the  other,  not  only  by 
their  shape  and  size,  but  also  by  the  peculiarity  of 
motility  which  some  of  them  were  seen  to  possess. 


INTR  OD  UCTION.  1 9 

In  the  year  1683  he  discovered  in  the  tartar  scraped 
from  between  the  teeth  a  form  of  micro-organism  upon 
which  he  laid  special  stress.  This  observation  he  em- 
bodied in  the  form  of  a  contribution  to  the  Royal  Society 
of  London  on  September  14,  1683.  This  paper  is  of 
peculiar  importance,  not  only  because  of  the  careful, 
objective  nature  of  the  description  given  of  the  bodies 
seen  by  him,  but  also  for  the  illustrations  which  accom- 
pany it.  From  a  perusal  of  the  text  and  an  inspection 
of  the  plates  there  remains  little  room  for  doubt  that 
Leeuwenhoek  saw  with  his  primitive  lens  the  bodies  now 
recognized  as  bacteria.1 

Upon  seeing  these  bodies  he  was  apparently  very 
much  impressed,  for  he  writes :  "  With  the  greatest 
astonishment  I  observed  that  everywhere  throughout 
the  material  which  I  was  examining  were  distributed 
animalcules  of  the  most  microscopic  dimensions,  which 
moved  themselves  about  in  a  remarkably  energetic  way." 

This  discovery  was  shortly  followed  by  others  of  an 
equally  important  nature.  His  field  of  observation 
appears  to  have  increased  rapidly,  for  after  a  time  he 
speaks  of  bodies  of  much  smaller  dimensions  than  those 
at  first  described  by  him. 

Throughout  all  of  Leeuwenhoek's  work  there  is  a 
conspicuous  absence  of  the  speculative.  His  contri- 
butions are  remarkable  for  their  purely  objective 
nature. 

After  the  presence  of  these  organisms  in  water,  in 
the  mouth,  and  in  the  intestinal  evacuations  was  made 
known  to  the  world,  it  is  not  surprising  that  they 
were  immediately  seized  upon  as  the  explanation  of  the 

1  See  Arcana  Naturae  detecta  ab  ANTONIO  VAN  LEEUWENHOEK  ; 
Delphis  Batavorum,  1695. 


.    ^BACTERIOLOGY. 

modern  med  cine.  Though  the  contributions  which 
have  done  most  to  place  bacteriology  on  the  footing  of 
a  science  are  those  of  recent  years,  still,  during  the 
earlier  stages  of  its  development,  many  observations 
were  made  which  formed  the  foundation-work  for  much 
that  was  to  follow.  Before  regularly  beginning  our 
studies,  therefore,  it  may  be  of  advantage  to  acquaint  our- 
selves with  the  more  prominent  of  those  investigations. 

Antony  van  Leeuwenhoek,  the  first  to  describe  the 
bodies  now  recognized  as  bacteria,  was  born  at  Delft,  in 
Holland,  in  1632.  He  was  not  considered  a  man  of 
liberal  education,  having  been  during  his  early  years  an 
apprentice  to  a  linendraper.  During  his  apprenticeship 
he  learned  the  art  of  lens-grinding,  in  which  he  became 
so  proficient  that  he  eventually  perfected  a  simple  lens 
by  means  of  which  he  was  enabled  to  see  objects  ^of 
much  smaller  dimensions  than  any  hitherto  seen  -fcith 
the  best  compound  microscopes  in  existence  at  that 
date.  At  the  time  of  his  discoveries  he  was  following 
the  trade  of  linendraper  in  Amsterdam. 

In  1675  he  published  the  fact  that  he  had  succeeded 
in  perfecting  a  lens  by  means  of  which  he  could  detect 
in  a  drop  of  rain-water  living,  motile  "animalcules" 
of  the  most  minute  dimensions — smaller  than  anything 
that  had  hitherto  been  seen.  Encouraged  by  this  dis- 
covery, he  continued  to  examine  various  substances  for 
the  presence  of  what  he  considered  animal  life  in  its 
most  minute  form.  He  found  in  sea-water,  in  well- 
water,  in  the  intestinal  canal  of  frogs  and  birds,  and  in 
his  own  diarrhoeal  evacuations,  objects  that  differenti- 
ated themselves  the  one  from  the  other,  not  only  by 
their  shape  and  size,  but  also  by  the  peculiarity  of 
motility  which  some  of  them  were  seen  to  possess. 


INTRODUCTION.  19 

In  the  year  1683  he  discovered  in  the  tartar  scraped 
from  between  the  teeth  a  form  of  micro-organism  upon 
which  he  laid  special  stress.  This  observation  he  em- 
bodied in  the  form  of  a  contribution  to  the  Royal  Society 
of  London  on  September  14,  1683.  This  paper  is  of 
peculiar  importance,  not  only  because  of  the  careful, 
objective  nature  of  the  description  given  of  the  bodies 
seen  by  him,  but  also  for  the  illustrations  which  accom- 
pany it.  From  a  perusal  of  the  text  and  an  inspection 
of  the  plates  there  remains  little  room  for  doubt  that 
Leeuwenhoek  saw  with  his  primitive  lens  the  bodies  now 
recognized  as  bacteria.1 

Upon  seeing  these  bodies  he  was  apparently  very 
much  impressed,  for  he  writes :  "  With  the  greatest 
astonishment  I  observed  that  everywhere  throughout 
the  material  which  I  was  examining  were  distributed 
animalcules  of  the  most  microscopic  dimensions,  which 
moved  themselves  about  in  a  remarkably  energetic  way." 

This  discovery  was  shortly  followed  by  others  of  an 
equally  important  nature.  His  field  of  observation 
appears  to  have  increased  rapidly,  for  after  a  time  he 
speaks  of  bodies  of  much  smaller  dimensions  than  those 
at  first  described  by  him. 

Throughout  all  of  Leeuwenhoek's  work  there  is  a 
conspicuous  absence  of  the  speculative.  His  contri- 
butions are  remarkable  for  their  purely  objective 
nature. 

After  the  presence  of  these  organisms  in  water,  in 
the  mouth,  and  in  the  intestinal  evacuations  was  made 
known  to  the  world,  it  is  not  surprising  that  they 
were  immediately  seized  upon  as  the  explanation  of  the 

1  See  Arcana  Naturse  detecta  ab  ANTONIO  VAN  LEEUWENHOEK  ; 
Delphis  Batavorum,  1695. 


modern  medicine.  Though  the  contributions  which 
have  done  most  to  place  bacteriology  on  the  footing  of 
a  science  are  those  of  recent  years,  still,  during  the 
earlier  stages  of  its  development,  many  observations 
were  made  which  formed  the  foundation-work  for  much 
that  was  to  follow.  Before  regularly  beginning  our 
studies,  therefore,  it  may  be  of  advantage  to  acquaint  our- 
selves with  the  more  prominent  of  those  investigations. 

Antony  van  Leeuwenhoek,  the  first  to  describe  the 
bodies  now  recognized  as  bacteria,  was  born  at  Delft,  in 
Holland,  in  1632.  He  was  not  considered  a  man  of 
liberal  education,  having  been  during  his  early  years  an 
apprentice  to  a  linendraper.  During  his  apprenticeship 
he  learned  the  art  of  lens-grinding,  in  which  he  became 
so  proficient  that  he  eventually  perfected  a  simple  lens 
by  means  of  which  he  was  enabled  to  see  objects-^f 
much  smaller  dimensions  than  any  hitherto  seen  4fcth 
the  best  compound  microscopes  in  existence  at  that 
date.  At  the  time  of  his  discoveries  he  was  following 
the  trade  of  linendraper  in  Amsterdam. 

In  1675  he  published  the  fact  that  he  had  succeeded 
in  perfecting  a  lens  by  means  of  which  he  could  detect 
in  a  drop  of  rain-water  living,  motile  "  animalcules " 
of  the  most  minute  dimensions — smaller  than  anything 
that  had  hitherto  been  seen.  Encouraged  by  this  dis- 
covery, he  continued  to  examine  various  substances  for 
the  presence  of  what  he  considered  animal  life  in  its 
most  minute  form.  He  found  in  sea-water,  in  well- 
water,  in  the  intestinal  canal  of  frogs  and  birds,  and  in 
his  own  diarrhoeal  evacuations,  objects  that  differenti- 
ated themselves  the  one  from  the  other,  not  only  by 
their  shape  and  size,  but  also  by  the  peculiarity  of 
motility  which  some  of  them  were  seen  to  possess. 


INTRODUCTION.  19 

In  the  year  1683  he  discovered  in  the  tartar  scraped 
from  between  the  teeth  a  form  of  micro-organism  upon 
which  he  laid  special  stress.  This  observation  he  em- 
bodied in  the  form  of  a  contribution  to  the  Royal  Society 
of  London  on  September  14,  1683.  This  paper  is  of 
peculiar  importance,  not  only  because  of  the  careful, 
objective  nature  of  the  description  given  of  the  bodies 
seen  by  him,  but  also  for  the  illustrations  which  accom- 
pany it.  From  a  perusal  of  the  text  and  an  inspection 
of  the  plates  there  remains  little  room  for  doubt  that 
Leeuwenhoek  saw  with  his  primitive  lens  the  bodies  now 
recognized  as  bacteria.1 

Upon  seeing  these  bodies  he  was  apparently  very 
much  impressed,  for  he  writes :  "  With  the  greatest 
astonishment  I  observed  that  everywhere  throughout 
the  material  which  I  was  examining  were  distributed 
animalcules  of  the  most  microscopic  dimensions,  which 
moved  themselves  about  in  a  remarkably  energetic  way." 

This  discovery  was  shortly  followed  by  others  of  an 
equally  important  nature.  His  field  of  observation 
appears  to  have  increased  rapidly,  for  after  a  time  he 
speaks  of  bodies  of  much  smaller  dimensions  than  those 
at  first  described  by  him. 

Throughout  all  of  Leeuwenhoek's  work  there  is  a 
conspicuous  absence  of  the  speculative.  His  contri- 
butions are  remarkable  for  their  purely  objective 
nature. 

After  the  presence  of  these  organisms  in  water,  in 
the  mouth,  and  in  the  intestinal  evacuations  was  made 
known  to  the  world,  it  is  not  surprising  that  they 
were  immediately  seized  upon  as  the  explanation  of  the 

1  See  Arcana  Naturse  detecta  ab  ANTONIO  VAN  LEEUWENHOEK  ; 
Del  phis  Batavorum,  1695. 


20  BACTERIOLOGY. 

origin  of  many  obscure  diseases.  So  universal  became 
the  belief  in  a  causal  relation  between  the  "animal- 
cules "  and  disease  that  it  amounted  almost  to  a  germ- 
mania.  It  became  the  fashion  to  suspect  the  presence 
of  these  organisms  in  all  forms  and  kinds  of  disease, 
simply  because  they  had  been  demonstrated  in  the 
mouth,  intestinal  evacuations,  and  water. 

Though  nothing  of  value  at  the  time  had  been  done 
in  the  way  of  classification,  and  even  less  in  separating 
and  identifying  the  members  of  this  large  group,  still 
the  foremost  men  of  the  day  did  not  hesitate  to  ascribe 
to  them  not  only  the  property  of  producing  pathological 
conditions,  but  some  even  went  so  far  as  to  hold  that 
variations  in  the  symptoms  of  disease  were  the  result 
of  differences  in  the  behavior  of  the  organisms  in  the 
tissues. 

Marcus  Antonius  Plenciz,  a  physician  of  Vienna  in 
1762,  declared  himself  a  firm  believer  in  the  work  of 
Leeuwenhoek,  and  based  the  doctrine  which  he  taught 
upon  the  discoveries  of  the  Dutch  observer  and  upon 
observations  of  a  confirmatory  nature  which  he  himself 
had  made.  The  doctrine  of  Plenciz  assumed  a  causal 
relation  between  the  micro-organisms  discovered  and 
described  by  Leeuwenhoek  and  all  infectious  diseases. 
He  maintained  that  the  material  of  infection  could  be 
nothing  else  than  a  living  substance,  and  endeavored  on 
these  grounds  to  explain  the  variations  in  the  period  of 
incubation  of  the  different  infectious  diseases.  He  like- 
wise believed  the  living  contagium  to  be  capable  of 
multiplication  within  the  body,  and  spoke  of  the  possi- 
bility of  its  transmission  through  the  air.  He  believed 
in  the  existence  of  a  special  germ  for  each  disease,  hold- 
ing that  just  as  from  a  given  cereal  only  one  kind  of 


INTRODUCTION.  21 

grain  can  grow,  so  by  the  special  germ  for  each  disease 
only  that  disease  can  be  produced. 

He  found  in  all  decomposing  matters  innumerable 
minute  "  animalculse,"  and  was  so  firmly  convinced  of 
their  etiological  relation  to  the  process  that  he  formu- 
lated the  law :  that  decomposition  can  only  take  place 
when  the  decomposable  material  becomes  coated  with  a 
layer  of  the  organisms,  and  can  proceed  only  when  they 
increase  and  multiply. 

However  convincing  the  arguments  of  Plenciz  may 
appear,  they  seem  to  have  been  lost  sight  of  in  the 
course  of  subsequent  events,  and  by  a  few  were  even 
regarded  as  the  productions  of  an  unbalanced  mind. 
For  example,  as  late  as  1820  we  find  Ozanam  express- 
ing himself  on  the  subject  as  follows  :  "  Many  authors 
have  written  concerning  the  animal  nature  of  the  con- 
tagion of  disease ;  many  have  indeed  assumed  it  to 
be  developed  from  animal  substances,  and  that  it  is 
itself  animal  and  possesses  the  property  of  life ;  I 
shall  not  waste  time  in  effort  to  refute  these  absurd 
hypotheses." 

Similar  expressions  of  opinion  were  heard  from  many 
other  investigators  of  the  time,  all  tending  in  the  same 
direction,  all  doubting  the  possibility  of  these  micro- 
scopic creatures  belonging  to  the  world  of  living  things. 

It  was  not  until  between  the  fourth  and  fifth  decades 
of  the  nineteenth  century  that  by  the  fortunate  coinci- 
dence of  a  number  of  important  discoveries  the  true  rela- 
tion of  the  lower  organisms  to  infectious  diseases  was 
scientifically  pointed  out.  With  the  fundamental  inves- 
tigations of  Pasteur  upon  the  souring  and  putrefaction  of 
beer  and  wine ;  with  the  discovery  by  Pollender  and 
Davaine  of  the  presence  of  rod-shaped  organisms  in  the 


22  BACTERIOLOGY. 

blood  of  animals  dead  of  splenic  fever,  and  with  the 
progress  of  knowledge  upon  the  parasitic  nature  of  cer- 
tain diseases  of  plants,  the  old  question  of  "  contagium 
animatum"  again  began  to  receive  attention.  It  was 
taken  up  by  Henle,  and  it  was  he  who  first  logically 
taught  this  doctrine  of  infection. 

The  main  point,  however,  that  had  occupied  the  atten- 
tion of  scientific  men  from  time  to  time  for  a  period  of 
about  two  hundred  years  subsequent  to  Leeuwenhoek's 
discoveries  was  the  origin  of  the  "  animalcules."  Do 
they  generate  spontaneously,  or  are  they  the  descendants 
of  pre-existing  creatures  of  the  same  kind  ?  was  the  all- 
important  question.  Among  the  earlier  participants 
in  this  discussion  were  many  of  the  most  distinguished 
men  of  the  day. 

In  1749  Needham,  who  held  firmly  to  the  opinion 
that  the  bodies  which  were  attracting  such  general  atten- 
tion developed  spontaneously  as  the  result  of  vegetative 
changes  in  the  substances  in  which  they  were  found, 
attempted  to  demonstrate  by  experiment  his  reasons  for 
holding  this  view.  He  maintained  that  the  bacteria 
which  appeared  about  a  grain  of  barley  germinating  in 
a  carefully  covered  watch-crystal  of  water  were  the 
result  of  changes  going  on  in  the  barley-grain  itself, 
incidental  to  its  germination. 

Spallanzani,  in  1769,  drew  attention  to  the  laxity  of 
Needham's  experimental  methods,  and  demonstrated  that 
if  infusions  of  decomposable  vegetable  matter  be  placed 
in  flasks,  which,  after  being  hermetically  sealed,  were 
heated  for  a  time  in  boiling  water,  no  living  organisms 
would  be  detected  in  them,  nor  would  decomposition 
appear  in  the  infusions  so  treated.  The  objection 
raised  by  Treviranus,  viz.,  that  the  high  temperature 


INTRODUCTION.  23 

to  which  the  infusions  had  been  subjected  had  so  al- 
tered them  and  the  air  about  them  that  the  conditions 
favorable  to  spontaneous  generation  no  longer  existed, 
was  promptly  met  by  Spallanzani  when  he  gently  tapped 
one  of  the  flasks  that  had  been  boiled  against  a  hard 
object  until  a  minute  crack  was  produced ;  invariably 
organisms  and  decomposition  appeared  in  the  flask  thus 
treated. 

From  the  time  of  the  experiments  of  Spallanzani 
until  as  late  as  1836  but  little  advance  was  made  in  the 
elucidation  of  this,  at  that  time,  obscure  problem. 

In  1836  Schulze  attracted  attention  to  the  subject  by 
the  convincing  nature  of  his  investigations.  He  showed 
that  if  the  air  which  gained  access  to  boiled  infusions 
be  robbed  of  its  living  organisms  by  first  passing  it 
through  strong  acid  or  alkaline  solutions  no  decom- 
position occurred,  and  living  organisms  could  not  be 
detected  in  the  infusions.  Following  quickly  upon 
this  contribution  came  Schwann,  in  1837,  and  somewhat 
later  (1854)  Schroder  and  Dusch,  with  similar  results 
obtained  by  somewhat  different  means.  Schwann  de- 
prived the  air  which  passed  to  his  infusions  of  its  living 
particles  by  conducting  it  through  highly  heated  tubes ; 
whereas  Schroder  and  Dusch,  by  means  of  cotton-wool 
interposed  between  the  boiled  infusions  and  the  outside 
air,  robbed  the  air  passing  to  the  infusions  of  its  organ- 
isms by  the  simple  process  of  filtration.  In  1860  Hoff- 
mann and  in  1861  Chevreul  and  Pasteur  demonstrated 
that  the  precautions  taken  by  preceding  investigators 
for  rendering  the  air  which  entered  these  flasks  free 
from  bacteria  were  not  necessary;  that  all  that  was 
required  to  prevent  the  access  of  bacteria  to  the  infu- 
sions in  the  flasks  was  to  draw  out  the  neck  of  the  flask 


24  BACTERIOLOGY. 

into  a  fine  tube,  bend  it  down  along  the  side  of  the 
flask,  and  then  bend  it  up  again  a  few  centimetres  from 
its  extremity,  and  leave  the  mouth  open.  The  infusion 
was  then  to  be  boiled  in  the  flask  thus  prepared  and  the 
mouth  of  the  tube  left  open.  The  organisms  which 
now  fell  into  the  open  end  of  the  tube  were  arrested  by 
the  drop  of  water  of  condensation  which  collected  at  its 
lowest  angle,  and  none  could  enter  the  flask. 

While,  from  our  modern  standpoint,  the  results  of 
these  investigations  seem  to  be  of  a  most  convincing 
nature,  yet  there  were  many  at  the  time  who  required 
additional  proof  that  "spontaneous  generation"  was 
not  the  explanation  for  the  mysterious  appearance 
of  these  minute  living  creatures.  The  majority,  if 
not  all,  of  such  doubts  were  subsequently  dissipated 
through  the  well-known  investigations  of  Tyndall  upon 
the  floating  matters  of  the  air.  In  these  studies  he 
demonstrated  by  numerous  ingenious  and  instructive 
experiments  that  the  presence  of  living  organisms  in 
decomposing  fluids  was  always  to  be  explained  either 
by  the  pre-existence  of  similar  living  forms  in  the  infu- 
sion or  upon  the  walls  of  the  vessel  containing  it,  or 
by  the  infusion  having  been  exposed  to  air  which  had 
not  been  deprived  of  its  viable  organisms. 

Throughout  all  the  work  bearing  upon  this  subject^ 
from  the  time  of  Spallanzani  to  that  of  Tyndall,  certain 
irregularities  were  constantly  appearing.  It  was  found 
that  particular  substances  required  to  be  heated  for  a 
much  longer  time  than  was  needed  to  render  other 
substances  free  from  living  organisms,  and  even  after 
the  most  careful  precautions  decomposition  would  occa- 
sionally occur. 

In   1762  Bonnet,  who  was  deeply  interested  in  this 


INTRODUCTION.  25 

subject,  suggested,  in  reference  to  the  results  obtained 
by  Needham,  the  possibility  of  the  existence  of  "  germs 
or  their  eggs,"  which  had  the  power  to  resist  the  tem- 
perature to  which  some  of  the  infusions  employed  in 
Needham's  experiments  had  been  subjected. 

More  than  a  hundred  years  after  Bonnet  had  in- 
dulged in  this  pure  speculation  it  became  the  happy 
privilege  of  Ferdinand  Cohn,  of  Breslau,  to  demonstrate 
its  accuracy  and  importance. 

Cohn  repeated  the  foregoing  experiments  with  like 
results.  He  concluded  that  the  irregularities  could 
only  be  due  to  either  the  existence  of  more  resistant 
species  of  bacteria  or  to  more  resistant  stages  into 
which  certain  bacteria  have  the  property  of  passing. 
He  demonstrated  that  some  of  the  rod-shaped  organ- 
isms possess  the  power  of  passing  into  a  resting-  or 
spore-stage  in  the  course  of  their  life-cycle,  analogous 
to  the  seeding  stage  of  higher  plants,  and  when  in 
this  stage  they  are  much  less  susceptible  to  the  dele- 
terious action  of  high  temperatures  than  when  they  are 
growing  as  normal  vegetative  forms.  With  the  discov- 
ery of  these  more  resistant  spores  the  doctrine  of  spon- 
taneous generation  received  its  death-blow.  It  was  no 
longer  difficult  to  explain  the  inconsistencies  in  the  re- 
sults of  former  investigations,  nor  was  it  any  longer  to  be 
doubted  that  putrefaction  and  fermentation  were  the  re- 
sult of  bacterial  life  and  not  the  cause  of  it,  and  that  these 
bacteria  were  the  offspring  of  pre-existing  similar  forms. 
In  other  words,  the  law  of  Harvey,  Omne  vivum  ex  ovo, 
or  its  modification,  Omne  vivum  ex  vivo,  was  shown  to 
apply  not  only  to  the  more  highly  organized  members 
of  the  animal  and  vegetable  kingdoms,  but  to  the  most 
microscopic,  unicellular  creatures  as  well. 


26  BACTERIOLOGY. 

The  establishment  of  this  point  gave  an  impetus  to 
further  investigations,  and  as  the  all-important  ques- 
tion was  that  concerning  the  relation  of  the  micro- 
scopic organisms  to  disease,  attention  naturally  turned 
into  this  channel  of  study.  Even  before  the  hypothesis 
of  spontaneous  generation  had  received  its  final  refutation 
a  number  of  observations  of  a  most  important  nature  had 
been  made  by  investigators  who  had  long  since  ceased  to 
consider  spontaneous  generation  as  a  tenable  explanation 
of  the  origin  of  the  microscopic  living  particles. 

In  the  main,  these  studies  had  been  conducted  upon 
wounds  and  the  infections  to  which  they  are  liable ;  in 
fact,  the  evolution  of  our  knowledge  of  bacteriology  to 
its  present  development  is  so  intimately  associated  with 
this  particular  line  of  investigation  that  a  few  historical 
facts  in  connection  with  it  may  not  be  without  interest. 

The  observations  of  Rindfleisch,  in  1866,  in  which 
he  describes  the  presence  of  small,  pin-head  points  in 
the  myocardium  and  general  musculature  of  individuals 
that  had  died  as  a  result  of  infected  wounds,  represent, 
probably,  the  first  reliable  contribution  to  this  subject. 
He  studied  the  tissue-changes  round  about  these  points 
up  to  the  stage  of  miliary  abscess-formation.  He  refers 
to  the  organisms  as  "  vibrios."  Almost  simultaneously 
von  Recklinghausen  and  Waldeyer  described  similar 
changes  that  they  had  observed  in  pyaemia  and,  occa- 
sionally, secondary  to  typhoid  fever.  Von  Reckling- 
hausen believed  the  granules  seen  in  the  abscess-points 
to  be  micrococci  and  not  tissue-detritus,  and  gave  as 
the  reason  that  they  were  regular  in  size  and  shape,  and 
gave  specific  reactions  with  particular  staining-fluids. 
Birch-Hirschfeld  was  able  to  trace  bacteria  found  in 
the  blood  and  organs  to  the  wound  as  the  point  of  en- 
trance, and  believed  both  the  local  and  the  constitutional 


INTRODUCTION.  27 

conditions  to  stand  in  direct  ratio  to  the  number  of  spher- 
ical bacteria  present  in  the  wound.  He  observed  also 
that  as  the  organisms  increased  in  number  they  could 
often  be  found  within  the  bodies  of  pus-corpuscles. 
His  studies  of  pyaemia  led  him  to  the  important  con- 
clusion that  in  this  condition  micro-organisms  were 
always  present  in  the  blood. 

Of  immense  importance  to  the  subject  were  the  in- 
vestigations of  Klebs,  made  at  the  Military  Hospital 
at  Carlsruhe  in  1870-?71.  He  not  only  saw,  as  others 
before  him  had  seen,  that  bacteria  were  present  in  dis- 
eases following  infection  of  wounds,  but  described  the 
manner  in  which  the  organisms  had  gained  entrance 
from  the  point  of  injury  to  the  internal  organs  and 
blood.  He  expressed  the  opinion  that  the  spherical 
and  rod-shaped  bodies  which  he  saw  in  the  secretions  of 
wounds  were  closely  allied,  and  he  gave  to  them  the 
designation  "  microsporon  septictim."  He  believed  that 
the  organisms  gained  access  to  the  tissues  round  about 
the  point  of  injury  both  by  the  aid  of  the  wandering 
leucocytes  and  by  being  forced  through  the  connec- 
tive-tissue lymph-spaces  by  the  mechanical  pressure  of 
muscular  contraction. 

On  erysipelatous  inflammations  secondary  to  injury 
important  investigations  were  also  being  made,  Wilde, 
Orth,  von  Recklinghausen,  Lukomsky,  Billroth,  Ehr- 
lich,  Fehleisen,  and  others  agreeing  that  in  these  con- 
ditions micro-organisms  could  always  be  detected  in  the 
lymph-channels  of  the  subcutaneous  tissues  ;  and  through 
the  work  of  Oertel,  Nassiloff,  Classen,  Letzerich,  Klebs, 
and  Eberth  the  constant  presence  of  bacteria  in  the 
diphtheritic  deposits  at  times  seen  on  open  wounds  was 
established. 


28  BACTERIOLOGY. 

Simple  and  natural  as  all  this  may  seem  to  us  now, 
the  stage  to  which  the  subject  had  developed  when  these 
observations  were  recorded  did  not  admit  of  their  meet- 
ing with  unconditional  acceptance.  The  only  strong 
argument  in  favor  of  the  etiological  relation  of  the 
organisms  that  had  been  seen  to  the  diseases  with  which 
they  were  associated  was  the  constancy  of  this  associa- 
tion. No  efforts  had  been  made  to  isolate  them,  and 
few  or  none  to  reproduce  the  pathological  conditions  by 
inoculation.  Moreover,  not  a  small  number  of  inves- 
tigators were  skeptical  as  to  the  importance  of  these 
observations  ;  many  claimed  that  micro-organisms  were 
normally  present  in  the  blood  and  tissues  of  the  body  ; 
and  some  even  urged  that  the  organisms  seen  in  dis- 
eased conditions  were  the  result  rather  than  the  cause 
of  the  maladies.  It  is  hardly  necessary  to  do  more 
than  say  that  both  of  these  views  were  purely  specula- 
tive, and  have  never  had  a  single  reliable  experimental 
argument  in  their  favor.  Billroth  and  Tiegel,  who  held 
to  the  former  opinion,  did  endeavor  to  prove  their  posi- 
tion through  experimental  means  ;  but  the  methods  em- 
ployed by  them  were  of  such  an  untrustworthy  nature 
that  the  fallacy  of  deductions  drawn  from  them  was 
very  quickly  made  manifest  by  subsequent  investigators. 
Their  method  for  demonstrating  the  presence  of  micro- 
organisms in  normal  tissues  was  to  remove  bits  of  organs 
from  the  healthy  animal  body  with  heated  instruments 
ivnd  drop  them  into  hot  melted  paraffin,  they  holding  that 
all  living  organisms  on  the  surface  of  the  tissues  would  be 
destroyed  by  the  high  temperature,  and  that  if  decom- 
position should  subsequently  occur  it  would  prove  that 
it  was  the  result  of  the  growth  of  bacteria  in  the  depths 
of  the  tissues  to  which  the  heat  had  not  penetrated. 


INTRODUCTION  2& 

Decomposition  did  usually  set  in,  and  they  accepted 
this  as  proof  of  the  accuracy  of  their  view.  Attention 
was,  however,  shortly  called  to  the  fact  that  in  cooling 
there  was  contraction  of  the  paraffin,  resulting  usually 
in  the  production  of  small  rents  and  cracks  in  which 
dust,  and  bacteria  lodged  upon  it,  could  accumulate  and 
finally  gain  access  to  the  tissues,  with  the  occurrence  of 
decomposition  as  a  consequence.  Their  results  were 
thus  explained  after  a  manner  analogous  to  that  em- 
ployed by  Spallanzani,  in  1769,  in  demonstrating  to 
Treviranus  the  fallacy  of  the  opinion  held  by  him  and 
the  accuracy  of  his  own  views,  viz.,  that  it  was  always 
through  the  access  of  organisms  from  without  that  de- 
composition primarily  originated.  (See  page  22.) 

Under  careful  precautions,  to  which  no  objection 
could  be  raised,  the  experiments  of  Billroth  and  Tiegel 
were  repeated  by  Pasteur,  Burdon-Sanderson,  and  Klebs, 
but  with  failure  in  every  instance  to  demonstrate  the 
presence  of  bacteria  in  the  healthy  living  tissues. 

The  fundamental  researches  of  Koch  (1881)  upon 
pathogenic  bacteria  and  their  relation  to  the  infectious 
diseases  of  animals  differed  from  those  of  preceding 
investigators  in  many  important  respects.  The  scien- 
tific methods  of  analysis  with  which  each  and  every 
obscure  problem  was  met  as  it  arose  served  at  once  to 
distinguish  him  as  a  pioneer  in  this  hitherto  but  imper- 
fectly cultivated  domain.  The  outcome  of  these  inves- 
tigations was  the  establishment  of  a  foundation  upon 
which  bacteriology  of  the  future  was  to  rest.  He,  for  the 
first  time,  demonstrated  that  distinct  varieties  of  infection, 
as  evidenced  by  anatomical  changes,  are  due  in  many 
cases  to  the  activities  of  specific  micro-organisms,  and 
that  by  proper  methods  it  is  possible  to  isolate  these 


30  BACTERIOLOGY. 

organisms  in  pure  culture,  to  cultivate  them  indefinitely 
under  artificial  conditions,  to  reproduce  the  lesions  by 
inoculation  of  these  pure  cultures  into  susceptible  ani- 
mals, and  to  continue  the  disease  at  will  by  continuous 
inoculation  from  an  infected  to  a  healthy  animal.  By 
the  methods  that  he  employed  he  demonstrated  a  series 
of  separate  and  distinct  diseases  that  can  be  produced  in 
mice  and  rabbits  by  the  injection  of  putrid  substances 
into  their  tissues.  The  disease  known  as  septicaemia  of 
mice ;  likewise  a  disease  characterized  by  progressive 
abscess-formation,  and  pyaemia  and  septicaemia  of  rab- 
bits, were  among  the  affections  first  produced  by  him  in 
this  way.  It  was  in  the  course  of  this  work  that  the 
Abbe  system  of  substage  condensing  apparatus  was  first 
used  in  bacteriology  ;  that  the  aniline  dyes  suggested  by 
Weigert  were  brought  into  general  use ;  that  the  isola- 
tion and  cultivation  of  bacteria  in  pure  culture  on  solid 
media  were  shown  to  be  possible;  and  that  animals 
were  employed  as  a  means  of  obtaining  from  mixtures 
pure  cultures  of  pathogenic  bacteria. 

With  the  bounteous  harvest  of  original  and  important 
suggestions  that  was  reaped  from  Koch's  classical  series 
of  investigations  bacteriology  reached  an  epoch  in  its 
development,  and  at  this  period  modern  bacteriolog) 
may  justly  be  said  to  have  had  its  birth. 

NOTE. — I  have  presented  only  the  most  prominent 
investigations  that  will  serve  to  indicate  the  lines  along 
which  the  subject  has  developed.  For  a  more  detailed 
account  of  the  historical  development  of  the  work  the 
reader  is  referred  to  Loffler's  instructive  and  enter- 
taining Vorlesungen  uber  die  geschichtliche  Entwickelung 
der  Lehre  von  den  Bacterien,  upon  which  I  have  drawn 
freely  in  preparing  the  foregoing  sketch. 


CHAPTER   I. 

Definition  of  bacteria — Differences  between  parasites  and  saprophytes 
— Their  place  in  nature — Bacterial  enzymes — Products  of  bacteria 
— Nutrition  of  bacteria — Their  relation  to  oxygen — Influence  of 
temperature  upon  their  growth — Chemotaxis. 

BACTERIA  (more  properly  bacteriacese  or  schizomy- 
cetesj  were  regarded  by  the  older  writers  as  infusoria. 
This  was  because  of  their  capacity  for  developing  in 
infusions,  their  property  of  spore-formation,  their  resist- 
ance to  drying,  their  power  of  independent  motion,  and 
the  absence  of  chlorophyll  from  their  tissues.  In  the 
modern  conception,  however,  this  classification  is  unten- 
able, and  bacteria,  by  virtue  of  their  distinguishing 
peculiarities,  are  now  treated  as  a  group  by  themselves 
that  may  briefly  be  defined  as  comprising  microscopic, 
unicellular,  vegetable  organisms  that  multiply  by  the 
process  of  transverse  division. 

Inasmuch  as  bacteria  are  not  possessed  of  chloro- 
phyll,1 their  metabolic  processes  are  fundamentally  dif- 
ferent from  those  of  the  higher  plants  in  which  it  is 
present.  They  cannot,  as  in  the  case  of  the  green 
plants,  obtain  carbon  and  nitrogen  from  such  simple 
bodies  as  carbon  dioxide  and  ammonia,  but  are  forced 
to  secure  these  essential  elements  from  organic  matter 
as  such.  This  power  to  decompose  and  assimilate 

1  Chlorophyll  is  the  green  coloring-matter  possessed  by  the  higher 
plants  by  means  of  which  they  are  enabled  in  the  presence  of  sunlight 
to  decompose  carbonic  acid  (COa)  and  ammonia  (NHs)  into  their  ele- 
mentary constituents. 

31 


32  BACTERIOLOGY. 

organic  matters  is  signally  different  in  different  species 
of  bacteria,  and,  singular  to  say,  there  is  a  small  group 
(to  be  described  later)  from  which  this  function  is  appar- 
ently absent,  in  spite  of  the  fact  that  no  compensatory 
chlorophyll  is  discernible  in  their  tissues. 

SAPROPHYTES  AND  PARASITES. — In  the  case  of 
certain  bacteria,  in  fact,  the  majority,  the  source  of  food- 
supply  must  of  necessity  be  dead  organic  matters  of  either 
animal  or  vegetable  origin.  They  cannot  exist  in  the 
presence  of  living  tissues.  To  the  members  of  this 
group  the  designation  saprophytic  or  metatrophic  (A. 
Fischer)  is  given.  To  that  group  that  can  exist  only 
upon  living  organic  matters,  and  herein  belong  many 
(not  all)  of  the  disease-producing  bacteria,  the  appella- 
tion parasitic  or  paratrop/iic  (A.  Fischer)  is  applied ; 
while  for  the  few  species  that  either  do  not  require 
organic  matters,  or  do  not,  so  far  as  is  known,  have  the 
faculty  of  decomposing  and  assimilating  proteid  stuffs 
at  all,  the  name  prototrophic  is  suggested  by  Fischer. 
In  the  strict  sense  of  the  word,  a  parasite  or  paratroph 
can  exist  only  in  the  body  of  a  living  host,  and  a  sapro- 
phyte or  metatroph  only  upon  lifeless  organic  matters, 
and  such  obligate  parasites  and  saphrophytes  are  known, 
but  in  the  majority  of  cases  such  nutritive  conditions  are 
not  obligatory,  many  of  both  metatrophs  and  paratroph s 
having  the  power  to  adapt  themselves  to  conditions  other 
than  those  for  which  they  are  by  nature  best  fitted.  For 
instance,  certain  species  that  exhibit  their  most  impor- 
tant properties  under  conditions  of  parasitism  may, 
nevertheless,  lead  a  metatrophic  existence  when  circum- 
stances demand  it,  and,  on  the  other  hand,  particular 
species  usually  metatrophic  by  nature  may  find  condi- 
tions favorable  to  their  development  in  a  living  host. 


THEIR  PLACE  IN  NATURE.  33 

To  such  adaptable  species  the  designation  "  facultative  " 
is  given,  and,  when  employed,  signifies  that  the  species 
in  question  has  the  faculty  of  adapting  itself  to  en- 
vironments other  than  those  in  which  it  is  usually  en- 
countered. In  this  sense  all  of  the  disease-producing 
bacteria  that  can  be  cultivated  artificially  are  manifestly 
facultative  metatrophs  or  saprophytes. 

The  life-processes  of  bacteria  are  so  rapid,  complex, 
and  energetic  that  they  result  in  the  most  profound 
alterations  in  the  structure  and  composition  of  the 
materials  in  and  upon  which  they  are  developing. 

Decomposition,  putrefaction,  and  fermentation  result 
from  the  activities  of  the  metatrophic  bacteria ;  while 
the  changes  brought  about  in  the  tissues  of  their  living 
host  by  the  purely  parasitic  forms  find  expression  in 
disease-processes,  and  not  infrequently  in  complete  death. 

THEIR  PLACE  IN  NATURE. — The  role  played  in 
nature  by  the  metatrophs  is  a  very  important  one. 
Through  their  functional  activities  the  highly  compli- 
cated tissues  of  dead  animals  and  vegetables  are  resolved 
into  the  simpler  compounds,  carbonic  acid,  water,  and 
ammonia,  in  which  form  they  may  be  taken  up  and  ap- 
propriated as  nourishment  by  the  more  highly  organized 
members  of  the  vegetable  kingdom.  It  is  through  this 
ultimate  production  of  carbonic  acid,  ammonia,  and 
water  by  bacteria,  as  end-products  in  the  processes  of 
decomposition  and  fermentation  of  dead  animal  and 
vegetable  tissues,  that  the  demands  of  growing  vegeta- 
tion for  these  compounds  are  supplied. 

The  chlorophyll  plants  do  not  possess  the  power  of 
obtaining  their  carbon  and  nitrogen  from  such  highly 
organized  and  complicated  substances  as  serve  for  the 
nutrition  of  bacteria,  and  as  the  production  of  the  simpler 
compounds,  carbon  dioxide  and  ammonia,  by  the  animal 
3 


34  BA  CTERIOL  OGY. 

world  is  not  sufficient  to  meet  the  demands  of  the  chlo- 
rophyll plants,  the  importance  of  the  part  played  by 
bacteria  in  making  up  this  deficit  cannot  be  overesti- 
mated. Were  it  not  for  the  activity  of  these  microscopic 
living  creatures  all  life  upon  the  surface  of  the  earth 
would  cease.  Deprive  higher  vegetation  of  the  carbon 
and  nitrogen  supplied  to  it  as  a  result  of  bacterial  ac- 
tivity, and  its  development  comes  rapidly  to  an  end ; 
rob  the  animal  kingdom  of  the  food-stuffs  supplied  to 
it  by  the  vegetable  world,  and  life  is  no  longer  pos- 
sible. It  is  plain,  therefore,  that  the  saprophytes,  which 
represent  the  large  majority  of  all  bacteria,  must  be 
looked  upon  in  the  light  of  benefactors,  without  which 
existence  would  be  impossible. 

With  the  parasites,  on  the  other  hand,  the  conditions 
are  far  from  analogous.  Through  their  metabolic  activ- 
ities there  is  constantly  a  loss,  rather  than  a  gain,  to  both 
the  animal  and  vegetable  kingdoms.  Their  host  must 
always  be  a  living  body  in  which  exist  conditions 
favorable  to  their  development,  and  from  which  they 
appropriate  substances  that  are  necessary  to  the  health 
and  life  of  the  organism  to  which  they  have  found 
access ;  at  the  same  time  they  eliminate  substances  as 
products  of  their  nutrition  that  are  directly  poisonous 
to  the  tissues  in  which  they  are  growing. 

In  their  relations  to  terrestrial  life,  the  positions  oc- 
cupied by  the  two  functionally  different  groups,  the  sap- 
rophytes on  the  one  hand,  and  the  parasites  on  the  other, 
are  diametrically  opposed  :  the  saprophytic  forms  stand- 
ing as  benefactors,  in  resolving  dead  animal  and  vege- 
table bodies  into  their  component  parts,  which  serve  as 
food  for  living  vegetation,  and  at  the  same  time  remov- 
ing from  the  surface  of  the  earth  the  remains  of  all  dead 


THEIR  PLACE  IN  NATURE.  35 

organic  substances ;  while  the  parasitic  group  exists 
only  at  the  expense  of  the  more  highly  organized  mem- 
bers of  both  the  animal  and  vegetable  kingdoms.  It  is 
to  the  parasitic  group  that  the  pathogenic1  organisms 
belong. 

In  addition  to  the  metatrophs  that  are  concerned  in 
the  changes  to  which  allusion  has  just  been  made,  there 
exist  other  species  whose  life-processes  result  in  specific 
changes  of  great  interest  and  importance.  Some  of 
these  are  characterized  by  their  property  of  producing 
pigments  of  different  color;  these  are  known  as  the 
chromogenic 2  species.  Just  what  the  biological  signifi- 
cance of  the  pigment-producing  function  is  cannot 
be  said,  but  the  fact  that  many  of  the  chromogens 
are  richly  endowed  with  proteolytic3  activity  makes  it 
probable  that  they  are,  in  common  with  other  meta- 
trophs, concerned  in  the  omnipresent  process  of  disinte- 
gration in  progress  in  all  dead  organic  matters,  and 
that,  after  all,  their  power  to  produce  colors,  though 
conspicuous,  is  of  but  subordinate  importance.  From 
the  investigations  of  Beyerinck  it  would  seem  that  in 
some  of  the  chromogenic  forms  the  pigment  is  an 
integral  part  of  the  bacteria  themselves ;  in  others  that 
it  is  an  excretory  product  of  species  that  are  them- 
selves colorless ;  while  in  still  others,  that  it  is  an 
excretory  product  which  remains  intimately  associated 
with  the  bacterial  cells  and  in  part  or  wholly  stains 
them. 

Others,  the  so-called  photogenic   or   phosphorescent 

1  Pathogenic  organisms  are  those  which  possess  the  property  of  pro- 
ducing disease. 

2 Chromogenic:  possessing  the  property  of  generating  color. 
3  Proteolytic  :  the  power  of  dissolving  or  digesting  proteids. 


36  BA  CTERIOL  OGY. 

bacteria,  possess  the  property  of  producing  light  or 
of  illuminating  the  medium  on  which  they  grow  by 
a  peculiar  phosphorescence.  These  are  found  in  sea- 
water  and  in  decomposing  phosphorescent  fish  and 
meat. 

1  Still  others,  the  so-called  zymogenic  bacteria,  are  con- 
cerned in  the  various  fermentations,  such,  for  instance, 
as  acetic-,  lactic-,  and  butyric-acid  fermentations  ;  and 
many  of  the  industries,  such,  for  example,  as  those 
concerned  in  the  making  of  wine,  beer,  cheese,  butter, 
and  indigo,  are  more  or  less  directly  dependent  upon 
the  fermentation  that  accompanies  the  growth  of  pecu- 
liar species  of  bacteria  in  those  materials. 

The  saprogenic  bacteria  are  those  that  produce  the 
particular  fermentation  that  we  know  as  putrefac- 
tion. 

Another  very  important  saprophytic  group  comprises 
the  so-called  nitrifying  and  denitrifying  bacteria,  whose 
activities  are  concerned  in  specific  forms  of  fermentation  : 
the  former  oxidizing  ammonia  to  nitrous  and  nitric  acids ; 
the  latter  reducing  nitric  acid  to  nitrous  acid  and  am- 
monia. It  is  through  their  association  (symbiosis)  with 
the  nitrifying  bacteria  that  certain  plants,  the  legumi- 
nous, are  enabled  to  make  up  their  nitrogen  deficit  in 
part  from  the  free  nitrogen  of  the  air.  The  discovery 
of  this  phenomenon  gave  to  free  atmospheric  nitro- 
gen a  biological  significance  that  had  hitherto  been 
denied  it. 

The  so-called  thiogenic  bacteria  convert  sulphuretted 
hydrogen  into  higher  sulphur  compounds. 

The  bacteria  concerned  in  the  foregoing  changes  per- 
form the  functions,  as  said,  by  virtue  of  special  fer- 
ments or  enzymes  that  are  elaborated  by  them. 


BACTERIAL  ENZYMES.  37 

BACTERIAL  ENZYMES. — The  enzymes  are,  in 'gen- 
eral, amorphous  products  of  living  protoplasm,  that 
are  able  to  split  up  large  quantities  of  complex  or- 
ganic and  inorganic  substances  into  smaller,  simpler, 
more  soluble,  and  diffusible  combinations.  Bacteria 
produce  a  variety  of  enzymes,  by  means  of  which 
they  are  able  to  derive  their  nutrition  from  complex 
molecular  substances.  The  enzymes  are  to  some  extent 
dialysable.  The  principal  enzymes  produced  by  bacteria 
are  proteolytic,  diastatic,  inverting,  coagulating,  and  sugar 
splitting. 

The  proteolytic  or  albumin-dissolving  enzymes  are 
formed  by  a  great  many  bacteria.  The  most  familiar 
indications  of  the  formation  of  a  proteolytic  enzyme  are 
seen  in  the  liquefaction  of  gelatin,  of  coagulated  blood- 
serum,  and  of  casein.  Most  frequently  the  proteolytic 
enzyme  is  allied  to  trypsin,  as  shown  by  Abbott  and 
Gildersleeve,1  since  the  liquefaction  or  digestion  pro- 
ceeds only  under  an  alkaline  reaction.  Some  bacteria, 
however,  produce  a  proteolytic  enzyme  analogous  to 
pepsin,  and  this  enzyme  is  active  under  an  acid  reac- 
tion. The  proteolytic  enzymes  of  different  bacteria 
vary  considerably  with  regard  to  their  resistance  to 
heat,  some  being  destroyed  in  a  few  minutes  when 
heated  to  60°  or  70°  C.,  while  others  may  be  boiled  for 
a  short  time  without  suffering  marked  deterioration  (see 
Abbott  and  Gildersleeve,  loc.  cit.).  The  proteolytic 
enzymes  also  differ  in  respect  to  their  susceptibility  to 
the  action  of  acids  and  other  chemicals. 

The  formation  of  proteolytic  enzymes  is  one  of  the 
functions  of  bacteria  that  is  easily  disturbed  by  external 
conditions.  Long-continued  cultivation  on  media  in 

1  Abbott  and  Gildersleeve :  Journ.  of  Med.  Eesearch,  vol.  v.,  1903. 


38  BACTERIOLOGY. 

which  this  function  is  not  required  may  lead  to  marked 
deterioration,  while  continued  cultivation  under  condi- 
tions calling  forth  this  function  may  result  in  the  pro- 
duction of  a  race  of  organisms  in  which  the  function  is 
unusually  prominent. 

The  addition  of  carbohydrates  and  of  glycerine  to 
culture-media  interferes  with  production  of  the  proteo- 
lytic  enzyme  by  many  species  of  bacteria,  as  shown  by 
Auerbach.1 

Diastatie  enzymes  convert  starch  into  sugar.  This 
function  is  best  studied  on  media  containing  starch,  as 
potato  infusion  or  solutions  of  starch.  By  appropriate 
tests  the  intermediate  steps  in  the  conversion  of  the 
starch  into  sugar  may  be  traced  by  testing  a  portion  of ' 
the  culture-medium  from  time  to  time.  Fermi 2  found 
this  function  in  a  large  number  of  bacteria  studied, 
especially  in  organisms  of  the  subtilis  group  and  in  the 
microspira  of  the  cholera  group. 

Inverting  enzymes  convert  saccharose  into  dextrose. 
These  enzymes  are  produced  by  comparatively  few 
bacteria.  Fermi  found  this  function  manifested  by 
bacillus  megatherium,  pseudomonas  fluorescens,  bacillus 
vulgaris,  microspira  comma,  microspira  Metchnikovi, 
and  others. 

Coagulating  enzymes  are  those  which  coagulate  milk. 
Rennet  may  be  taken  as  the  typical  form.  This  altera- 
tion is  quite  common  in  association  with  an  acid  reac- 
tion, but  in  such  instances  it  is  not  always  certain  that 
the  coagulation  has  not  been  induced  by  the  acid  formed. 
Gorini3  found  that  cultures  of  bacillus  prodigiosus,  ster- 

1  Auerbach :  Archiv  fur  Hygiene,  Bd.  xxxi.  p.  311. 
»2  Fermi:  Archiv  fur  Hygiene,  Bd.  xi.,  and  Centralblatt  fur  Bacte- 
riologie,  Bd.  xii. 
3  Qorini :  Centralblatt  fur  Bacteriologie,  Bd.  xii.  p.  666. 


BACTERIAL  ENZYMES.  39 

ilized  by  heating  to  60°  C.,  caused  a  solid  coagulation  of 
sterile  milk  in  a  few  days. 

A  small  number  of  bacteria  have  also  been  encoun- 
tered that  bring  about  coagulation  of  milk  with  a  dis- 
tinctly alkaline  reaction.  This  function  has  been 
noticed  in  bacteria  isolated  from  milk,  and  especially 
in  bacterium  pseudodiphtheriticum  isolated  from  cows7 
milk  (Bergey). 

Sugar-splitting  enzymes  are  very  common  in  bacteria. 
This  function  varies  in  different  species  as  seen  in  the 
different  end-products  that  are  formed.  Buchner  suc- 
ceeded in  isolating  the  sugar-splitting  enzyme  (zymase) 
of  yeast-cells,  and  when  thus  isolated  it  still  possesses 
the  power  of  inducing  active  fermentation  of  sugar.  It 
is  believed  that  the  sugar-splitting  enzymes  of  bacteria 
are  similar  in  character  to  the  zymase  of  yeast-cells. 
The  splitting  up  of  carbohydrates  appears  to  be  brought 
about  by  the  bacteria  for  the  purpose  of  obtaining  oxygen, 
as  indicated  by  the  nature  of  the  end-products  formed, 
and  also  by  the  conditions  under  which  it  may  be  car- 
ried out — i.  e.,  the  absence  of  atmospheric  oxygen. 

The  splitting  of  the  carbohydrate  molecule  may  be 
illustrated  as  follows  : 

C6H1206  =  2C2H60  +  2C02 
Grape  sugar  —  2  alcohol  -\-  2  carbon  dioxid. 

or  C6H1206  =  2C3H603 

Grape  sugar  =  2  lactic  acid. 

or  C6H1206  =  3C.H  A 

Grape  sugar  —  3  acetic  acid. 

According  to  Theobald  Smith  !  all  facultative  anaero- 
bic bacteria  form  acids  from  carbohydrates,  while  the 
strict  aerobic  bacteria   do    not    have    this    function,  or 
'Theobald  Smith  :  Centralblatt  fur  Bacteriologie.  Bd.  xviii. 


40  BACTERIOLOGY. 

bring  about  the  alteration  so  slowly  that  it  is  concealed 
by  the  simultaneous  production  of  alkali.  Among  the 
acids  formed  by  bacteria,  besides  carbon  dioxid,  we  have 
lactic,  acetic,  butyric,  proprionic,  and  formic ;  and  fre- 
quently there  is  also  produced  ethyl  alcohol,  aldehyd, 
and  acetone. 

The  lactic  acid  formed  by  the  action  of  different  bac- 
teria on  carbohydrates  may  be  either  dextrorotatory  or 
laevorotatory,  or  almost  equal  quantities  of  both  forms 
may  be  present  and  the  mixture  be  optically  inactive. 

PRODUCTS  OF  BACTERIA. — As  stated,  bacteria  that 
produce  disease  are  known  as  pathogenic.  They  induce 
disease  by  their  poisonous  action  upon  the  tissues  in 
which  they  are  located.  The  materials  of  which  cer- 
tain species  of  bacteria  are  constructed,  and  the  products 
of  growth  of  certain  others,  are  of  the  greatest  import- 
ance in  their  relation  to  animal  and  human  pathology. 
Particular  species,  while  not  eliminating  soluble  poisons 
as  a  product  of  metabolism,  are  nevertheless  themselves 
built  up  of  poisonous  proteids,  or  of  proteids  with 
which  toxic  materials  are  so  intimately  associated  that 
they  can  only  be  isolated  by  the  most  refined  and  elabo- 
rate chemical  manipulations.  Others  produce  in  the 
course  of  their  growth  soluble  poisons  that  can  readily 
be  separated,  by  very  simple  methods,  from  the  bacteria 
that  produced  them.  The  proteid  matters  making  up 
the  bodies  of  many  species  of  bacteria,  even  those  -not 
conspicuously  pathogenic,  have  been  shown  by  Buchner 
to  induce  disease  when  isolated  and  injected  into  the 
tissues  of  animals ;  in  some  cases  causing  only  rise  of 
body-temperature,  in  others  acute  inflammatory  proc- 
esses with  pus-formation.  To  such  proteids  Buchner 
has  given  the  name  bacterial  proteins. 


NUTRITION  OF  BACTERIA.  41 

The  poisonous  soluble  products  of  bacterial  growth 
are  known  as  toxins  and  ptomains :  toxins  being,  in  gen- 
eral, uncrystallizable  poisons,  secreted  or  excreted  by 
the  bacteria,  of  whose  intimate  chemical  nature  little  or 
nothing  is  known ;  while  ptomains  are  crystallizable prod- 
ucts of  their  metabolic  activity  which,  physically  speaking, 
are  analogous  to  the  ordinary  vegetable  alkaloids. 

NUTRITION  OF  BACTERIA. — We  have  said  that 
through  the  agency  of  chlorophyll,  in  the  presence  of 
sunlight,  the  green  plants  are  enabled  to  obtain  the 
amount  of  nitrogen  and  carbon  which  is  necessary  to 
their  growth  from  such  simple  bodies  as  carbon  dioxide 
and  ammonia,  which  they  decompose  into  their  ele- 
mentary constituents.  The  bacteria,  on  the  other  hand, 
owing  to  the  absence  of  chlorophyll  from  their  tissues, 
do  not  possess  this  power.  They  must,  therefore,  have 
their  carbon  and  nitrogen  presented  as  such,  in  the  form 
of  decomposable  organic  substances. 

In  general,  bacteria  obtain  their  nitrogen  most 
readily  from  soluble  albumins,  and  to  a  certain  extent, 
but  by  no  means  so  easily,  from  salts  of  ammonium.  In 
some  of  Niigeli's  experiments  it  appeared  probable  that 
they  could  obtain  the  necessary  amount  of  nitrogen 
from  inorganic  nitrates.  At  all  events,  he  was  able 
in  certain  cases  to  demonstrate  a  reduction  of  nitric  to 
nitrous  acid  and  ultimately  to  ammonia.  Neverthe- 
less, in  all  of  these  experiments  circumstances  point  to 
the  probability  that  the  nitrogen  obtained  by  the  bac- 
teria for  building  up  their  tissues  in  the  course  of 
their  development  was  derived  from  some  source 
other  than  the  nitric  acid  or  the  nitrates,  and  that  the 
reduction  of  this  acid  was  most  probably  a  secondary 
phenomenon.  It  must  be  borne  in  mind,  however,  that 


42  BACTERIOLOGY. 

there  exists  a  specific  group  of  bacteria,  the  nitrifying 
bacteria,  that  apparently  increase  and  multiply  without 
appropriating  proteid  nutrition.  They  are  concerned 
in  the  particular  form  of  fermentation  that  results  in 
the  oxidation  of  ammonia  to  nitrous  and  nitric  acids,  a 
process  everywhere  in  progress  in  the  superficial  layers 
of  the  soil. 

For  the  supply  of  carbon  many  of  the  carbon  com- 
pounds serve  as  sources  upon  which  the  bacteria  can 
draw.  The  carbon  deficit,  for  example,  can  be  obtained 
from  sugar  and  bodies  of  like  composition  ;  from  glyc- 
erin and  many  of  the  fatty  acids ;  and  from  the  alka- 
line salts  of  tartaric,  citric,  malic,  lactic,  and  acetic 
acids.  In  some  instances  carbon  compounds,  which 
when  present  in  concentrated  form  inhibit  the  growth 
of  bacteria,  may,  when  highly  diluted,  serve  as  nutri- 
tion for  them.  Salicylic  acid  and  ethyl  alcohol  are  of 
this  class. 

In  addition  to  carbon  and  nitrogen,  water  is  essential 
to  the  life  and  development  of  bacteria.  Without  it 
no  development  occurs,  and  in  many  cases  drying  the 
organisms  results  in  their  death.  Certain  species  and 
developmental  forms,  on  the  contrary,  though  incapable 
of  multiplying  when  in  the  dry  state,  may  be  com- 
pletely deprived  of  their  water  without  causing  them 
to  lose  the  power  of  reproduction  under  favorable  con- 
ditions. 

Closer  study  of  bacteria,  and  a  more  intimate  ac- 
quaintance with  their  nutritive  changes,  demonstrate 
an  appreciable  variability  in  the  character  of  the  sub- 
stances best  suited  for  the  nutrition  of  different  species, 
one  requiring  a  tolerably  concentrated  form  of  nutri- 
tion, while  another  needs  but  a  very  limited  amount 


NUTRITION  OF  BACTERIA.  43 

of  proteid  substance  for  its  development.  Certain 
of  them  bring  about  most  profound  alterations  in  the 
media  in  which  they  exist,  while  others  produce  but 
little  apparent  change.  In  one  case  alterations  in  the 
reaction  of  the  media  will  be  conspicuous,  while  in 
another  no  such  variation  can  be  detected.  With  the 
growth  of  some  forms  products  resulting  from  specific 
processes  of  fermentation  appear.  Other  varieties  pro- 
duce poisons  of  remarkable  degrees  of  toxicity,  while  the 
growth  of  others  may  be  accompanied  by  the  evolution 
of  compounds  characteristic  of  putrefaction. 

In  considering  the  normal  development  of  bacteria 
we  must  not  lose  sight  of  the  fact  that  this  is  influenced 
both  by  the  quality  and  the  quantity  of  the  nutritive  mate- 
rials to  which  they  have  access,  and  by  the  character  of 
the  metabolic  products  that  accumulate  in  these  materials 
as  a  result  of  their  vital  processes.  Nitrogen  and  carbon 
compounds  may  be  present  in  amount  and  kind  entirely 
suitable  to  normal  bacterial  growth,  and  yet  this  may 
be  checked,  after  a  comparatively  short  time,  by  the 
accumulated  products  of  bacterial  metabolism,  some  of 
which  possess  the  property  of  inhibiting  growth  and 
ultimately  of  even  destroying  the  bacteria  that  produced 
them.  The  most  common  and  conspicuous  examples 
of  the  inhibiting  conditions  that  are  coincident  with 
bacterial  growth  are  alterations  in  the  chemical  reaction 
of  the  matters  in  which  the  bacteria  are  developing.  Since 
the  majority  of  them  grow  best  in  media  of  a  neutral  or 
very  slightly  alkaline  reaction,  any  excessive  development 
of  alkalinity  or  acidity,  as  a  result  of  growth,  arrests 
development,  and  no  evidence  of  life  or  further  multi- 
plication can  be  detected  until  this  deviation  from  the 
neutral  (or  the  suitable)  reaction  has  been  corrected. 


44  BACTERIOLOGY. 

THEIR  RELATION  TO  OXYGEN. — Of  considerable 
importance  and  interest  in  the  study  of  the  nutritive 
changes  of  bacteria  is  the  difference  in-  their  relation  to 
oxygen.  For  certain  species  free  oxygen  is  essential  to 
the  proper  performance  of  their  functions ;  in  another 
group  no  evidence  of  life  can  be  detected  under  its 
access ;  while  in  a  third  group  free  oxygen  appears  to 
play  but  an  unimportant  role,  for  development  occurs 
as  well  with  as  without  it,  It  was  Pasteur  who  first, 
demonstrated  the  existence  of  particular  species  of  bac- 
teria which  not  only  grow  and  multiply  and  perform 
definite  physiological  functions  without  the  aid  of  free 
oxygen,  but  to  the  existence  of  which  it  is  positively 
harmful.  To  these  he  gave  the  name  anaerobic  bac- 
teria, in  contradistinction  to  the  aerobic  group,  for  the 
proper  performance  of  whose  functions  free  oxygen  is 
essential.  The  anaerobic  bacteria  derive  their  oxygen 
entirely  from  oxygen  compounds  in  the  materials  in 
which  they  are  growing.  In  addition  to  these  there  is  a 
third  group,  for  the  maintenance  of  whose  existence  the 
absence  or  presence  of  uncombined  oxygen  is  apparently 
of  no  moment — development  progresses  as  well  with  as 
without  it ;  the  members  of  this  group  comprise  the  class 
known  as  facultative  in  their  relation  to  this  gas.  It  is 
to  this  third  group,  the  facultative,  that  the  majority  of 
bacteria  belong.  Since  all  growing  bacteria,  anaerobic 
as  well  as  aerobic,  generate  carbonic  acid  in  the  course 
of  their  development,  it  is  evident  that  oxygen  must 
in  reality  be  obtained  by  them  from  some  source,  and 
must  be  regarded  as  essential  to  their  life-processes; 
but -the  manner  in  which  it  is  appropriated  by  them 
varies,  the  aerobic  species  taking  it  from  the  air  as  free 
oxygen,  while  the  anaerobic  species,  not  possessed  of 


INFLUENCE  OF  TEMPERATURE  UPON  GROWTH.   45 

this  power,  obtain  it  through  the  decomposition  of  more 
or  less  stabfe  oxygen-containing  compounds. 

Though  the  multiplication  of  the  facultative  varieties 
is  not  interfered  with  by  either  the  presence  or  absence 
of  free  oxygen,  yet  experiments  demonstrate  that  the 
products  of  their  growth  are  different  under  the  varying 
conditions  of  absence  or  presence  of  this  gas.  For  ex- 
ample :  in  the  case  of. certain  of  the  chromogenic  forms 
the  presence  or  absence  of  oxygen  has  a  very  decided 
x  effect  .upon  the  production  of  the  pigments  by  which 
they  are  characterized. 

NOTE. — Observe  the  difference  between  the  intensity 
of  color  produced  upon  the  surface  of  the  medium  and 
that  along  the  track  of  the  needle  in  stab-cultures  of 
bacillus  prodigiosus  and  of  spirillum  rubrum.  In  the 
former  the  red  color  is  apparently  a  product  dependent 
upon  the  presence  of  oxygen,  while  in  the  latter  the 
greatest  intensity  of  «color  occurs  at  the  point  furthest 
removed  from  the  action  of  oxygen. 

INFLUENCE  OF  TEMPERATURE  UPON  THE  GROWTH. 
—Another  factor  which  plays  a  highly  important  part 
in  the  biological  functions  of  these  organisms  is  the 
temperature  under  which  they  exist.  The  extremes  of 
temperature  between  which  the  majority  of  bacteria  are 
known  to  grow  range  from  5.5°  to  43°  C.  At  the 
former  temperature  development  is  hardly  appreciable  ; 
it  becomes  more  and  more  active  until  38°  C.  is  reached, 
when  it  is  at  its  optimum,  and,  as  a  rule,  ceases  at 
43°  C. ;  though  species  exist  that  multiply  at  as  liigh 
a  temperature  as  70°  C.  and  others  at  as  low  as  0°  C, 


46  BACTERIOLOGY. 

The  investigations  of  Globig,1  Miquel,2  and  Macfadyen 
and  Bloxall3  have  revealed  the  existence  in  the  soil, 
in  water,  in  fa3ees,  in  sewage,  in  dust,  and,  in  fact,  prac- 
tically everywhere,  of  bacteria  that  under  artificial  culti- 
vation show  no  evidence  of  life  at  a  temperature  lower 
than  60°  to  65°  C.,  and  will  even  grow  at  such  high 
temperatures  as  70°  and  75°  C.,  a  state  of  affairs  almost 
paradoxical,  inasmuch  as  these  are  temperatures  that  suf- 
fice for  the  coagulation  of  albumin,  and,  in  consequence, 
are  generally  incompatible  with  life.  Rabinowitsch4 
has  likewise  described  a  number  of  species  of  these 
thermophilic  bacteria,  as  they  are  called ;  but  states  that 
it  was  possible  in  her  experiments  to  obtain  evidence 
of  their  growth  at  a  lower  temperature  (34°  to  44°  C.), 
as  well  as  at  the  higher  temperature  mentioned  by  pre- 
ceding investigators. 

The  most  favorable  temperature  for  the  development 
of  pathogenic  bacteria  is  that  of  the  human  body,  viz., 
37.5°  C.  There  are  a  number  of  bacteria  commonly 
present  in  water,  the  so-called  normal  water  bacteria., 
that  grow  best  at  about  20°  C. 

Under  natural  conditions  it  frequently  occurs  that 
the  development  of  one  species  or  group  of  species  of 
bacteria  is  directly  dependent  upon  the  functional  ac- 
tivities of  another  totally  distinct  species,  the  growth  of 
one  group  resulting  in  conditions  that  are  of  vital  im- 
portance to  the  existence  of  the  other.  This  interde- 
pendence of  species  is  known  as  symbiosis.  It  is  observed, 
for  instance,  in  the  course  of  putrefaction,  where, 

1  Globig :  Zeitschrift  fur  Hygiene,  Bd.  iii.  S.  294. 

2  Miquel :  Annales  de  Micrographie,  1888,  pp.  4  to  10. 

3  Macfadyen  and  Bloxall :  Journal  of  Path,  and  Bact.,  vol.  iii.  part  i. 

4  Rabinowitsch :  Zeitschrift  fiir  Hygiene  u.  lufectiouskrankheiten, 
Bd.  xx.  Heft  1,  S.  154  to  164. 


CHEMOTAX1S.  47 

through  exhaustion  of  free  oxygen  by  the  actively 
germinating  aerobic  varieties,  the  conditions  are  sup- 
plied that  enable  the  anaerobic  species  to  develop  and 
exercise  their  biological  activities.  Again,  through  the 
proteolytic  activity  of  enzymes  produced  by  certain 
species  of  bacteria,  other  species  are  supplied  with  nu- 
trition that  would  otherwise  be  unassimilable  or  only 
imperfectly  so.  Similar  symbiotic  relations  betAveen 
bacteria  and  higher  plants  are  also  noticed,  notably  that 
between  certain  bacteria  of  the  soil  and  the  group  of 
leguminous  plants,  whereby  the  latter  are  enabled, 
through  the  assistance  of  the  former,  to  make  up  their 
nitrogen  deficit  in  large  part  from  the  free  nitrogen  of 
the  atmosphere.  (See  page  36.) 

CHEMOTAXIS. — Another  interesting  biological  pecu- 
liarity of  bacteria  is  that  discovered  by  Engelmann  and 
by  Pfeffer,  known  as  chemotaxis.  This  term  applies  to 
the  peculiar  phenomena  of  attraction  and  of  repulsion 
that  are  exhibited  by  motile  bacteria  when  in  the  pres- 
ence of  solutions  of  bodies  of  various  chemical  compo- 
sition. Engelmann  demonstrated  that  the  bacteria  in 
decomposing  infusions  accumulate  in  great  numbers  in 
the  neighborhood  of  the  sources  of  oxygen.  In  a  hang- 
ing-drop of  such  an  infusion  the  bacteria  will  be  seen  to 
accumulate  in  a  dense  mass  along  the  edge  or  around  the 
edge  of  small  bubbles  of  air  in  the  fluid.  Even  plant 
cells  in  the  infusion,  whose  chlorophyll  sets  free  oxygen 
in  the  light,  are  surrounded  by  large  numbers  of  bacteria. 
The  positive  chemotactic  affinity  between  oxygen  and  bac- 
teria was  employed  by  Engelmann  as  a  basis  for  the  dem- 
onstration of  small  quantities  of  oxygen  in  studying  the 
assimilative  action  of  various  kinds  of  light  upon  the 
green  plant-cell.  Pfeffer  showed  that  when  a  neu- 


48  BACTERIOLOGY. 

tral  fluid  (a  drop  of  water)  containing  motile  bacteria 
is  brought  in  contact  with  a  weak  solution  of  either 
peptone,  sodium  chloride,  or  dextrin,  the  bacteria  are  at 
once  attracted  toward  the  solution ;  this  reaction  is 
designated  "  positive  chemotaxis."  On  the  other  hand, 
if  brought  in  contact  with  an  acid,  an  alkaline,  or  an 
alcoholic  solution,  the  bacteria  are  repelled  or  driven 
from  the  point  at  which  the  two  fluids  are  diffusing; 
that  is,  they  exhibit  "  negative  chemotactic "  affinities. 
The  significance  of  these  reactions  is  not  understood, 
but  it  has  been  aptly  suggested  that  they  may  be  funda- 
mentally analogous  to  the  specific  positive  and  negative 
affinities  exhibited  by  the  ions  (see  page  91)  resulting 
from  the  dissociation  of  electrolytes,  and  that  they  may 
"  have  their  explanation  in  the  forces  of  ionic  attraction 
and  repulsion." l  In  this  connection  it  is  important  to 
note  that  the  wandering  cells  of  the  animal  body,  the 
leucocytes,  exhibit  also  these  chemotactic  phenomena; 
and  it  is  especially  necessary  to  a  complete  comprehen- 
sion of  the  process  of  suppuration  to  bear  in  mind  that 
among  the  substances  which  have  the  greatest  attraction 
for  these  wandering  cells,  are  the  products  of  growth  of 
certain  bacteria  in  some  cases,  and  the  protoplasmic  con- 
stituents of  the  bacteria  themselves  in  others. 

From  what  has  been  learned,  it  may  be  said,  in 
general,  that  for  the  growth  and  development  of  bacteria 
organic  matter  of  a  neutral  or  slightly  alkaline  reaction, 
in  the  presence  of  moisture  and  at  a  suitable  temperature, 
is  all  that  is  necessary.  From  this  can  be  formed  some  idea 
of  the  omnipresence  in  nature  of  these  minute  vegetables. 
Bacteria  maybe  found  wherever  these  conditions  obtain. 

1  Read  Sewall  on  "Some  Relations  of  Osmosis  and  Ionic  Action  in 
Clinical  Medicine,"  International  Clinics,  vol.  xi.,  Eleventh  Series. 


CHAPTER    II. 

Morphology  l  of  bacteria — Chemical  composition  of  bacteria — Classi- 
fication of  bacteria  into  families  and  genera — Grouping — Mode  of 
multiplication — Spore-formation — Motility— The  thermal  death  - 
point  of  bacteria. 

IN  structure  the  bacteria  are  unicellular,  always  de- 
veloping from  pre-existing  cells  of  the  same  character 
and  never  appearing  spontaneously.  They  are  seen  to 
occur  as  spherical,  rod-  and  spiral-shaped  bodies  that 
multiply  by  the  simple  process  of  transverse  division, 
belonging,  therefore,  to  the  schizomycdes  or  fission 
fungi. 

Under  what  we  are  accustomed  to  regard  as  normal 
conditions  of  development,  and  by  the  ordinary  methods 
of  examination,  bacteria  appear  very  simple  in  form  and 
structure.  They  are  cells  consisting  of  a  protoplasmic 
mass  within  a  membranous  hull  that  is  discernible  with 
more  or  less  difficulty.  The  protoplasmic  body  is  of 
material  closely  allied,  chemically  speaking,  to  ordinary 
vegetable  proteid.  It  is  often  homogeneous,  but  in  par- 
ticular species  and  under  various  conditions  of  growth 
the  central  mass  in  stained  specimens  is  commonly 
marked  by  the  presence  of  very  dark  granules,  the 
so-called  metachromatic  granulations.  Again,  in  other 
species  paraplastic  granules  giving  the  microchemical 
reactions  of  fat,  starch,  sulphur,  etc.,  are  to  be  seen. 
Under  certain  physical  conditions  the  protoplasmic  body 
presents  irregular  rents  or  retractions,  the  result  of  pro- 
teolytic  or  of  osmotic  disturbances  dependent  upon  the 

1  Morphology :  pertaining  to  shape,  outline,  structure. 
4  49 


50  BACTERIOLOGY. 

character  of  the  fluid  in  which  the  bacteria  are  located  ;  in 
fact,  the  deeply  staining  grannies,  other  than  those  of  fat, 
starch,  and  sulphur,  that  are  often  observed,  are  regarded 
by  some  writers  (especially  A.  Fischer)  as  but  altered  or 
condensed  protoplasm  due  to  the  same  influences. 

In  certain  species  the  protoplasmic  body  is  always 
more  dense  at  the  poles  of  the  cells  than  at  the  middle, 
so  that  when  stained  the  ends  are  much  darker  than  the 
intervening  portion.  Sometimes  in  other  species  the 
reverse  is  the  case. 

By  some  investigators  the  protoplasmic  central  mass 
is  regarded  as  a  nucleus,  and,  functionally  speaking, 
possibly  it  is  to  all  intents  and  purposes,  but  this  cannot 
be  certainly  decided.  In  the  great  majority  of  cases, 
however,  with  the  ordinary  methods  of  examination,  it 
is  not  seen  to  possess  any  of  the  structural  peculiarities 
that  we  are  accustomed  to  regard  as  the  distinguishing 
attributes  of  cell-nuclei. 

The  enveloping  hull  or  membrane  is  in  some  cases 
apparently  only  a  modification  of  the  protoplasmic  cen- 
tral mass,  at  times  being  only  a  condensation  of  that 
protoplasm ;  again,  it  seems  to  be  chemically  different 
from  it.  In  a  few  instances  it  appears  to  be  allied  to 
cellulose  in  its  chemical  composition.  Sometimes  it  is 
so  thick  as  to  be  readily  seen,  while  again  it  is  discerni- 
ble only  by  special  methods  of  examination.  In  partic- 
ular species  it  may,  by  appropriate  methods,  be  seen  as 
a  sharply  defined  capsule  inclosing  a  clear  zone  in  which 
the  deeply  stained  central  mass  lies.  Occasionally  the 
central  protoplasmic  mass  is  surrounded  by  an  ill- 
defined  slimy  material  that  causes  the  individual  cells 
to  adhere  to  one  another  in  more  or  less  compact  masses 
or  pellicles  (zoogloea,  Fig.  1), 


CHEMICAL   COMPOSITION  OF  BACTERIA.         51 

CHEMICAL  COMPOSITION  OF  BACTERIA. — The  bodies 
of  bacteria  consist  of  water,  salts,  and  albuminous  sub- 
stances, with  smaller  proportions  of  various  extractive 
substances  soluble  in  alcohol  or  ether,  such  as  triolein, 
tripalmitin,  tristearin,  lecithin,  and  cholesterin.  In 
many  varieties  substances  giving  the  reaction  of  starch 
have  been  found,  while  others  give  the  true  reactions 
of  cellulose  (B.  subtilis).  Nuclein  has  not  been  found 
in  any  of  the  bacteria,  though  the  nuclein  bases,  xan- 
thin,  guanin,  adenin,  have  been  found. 

The  relative  amounts  of  water  in  bacteria  are  influ- 
enced to  a  large  extent  by  the  nature  of  the  medium  on 


Zooglcea  of  bacilli. 

which  they  have  been  grown.  In  like  manner  the  con- 
tent in  albumin,  extractive  substances,  and  salts  varies 
with  the  conditions  under  which  the  bacteria  have  been 
cultivated.  E.  Cramer1  has  studied  the  chemical  com- 
position of  bacteria  in  great  detail.  As  the  result  of 
his  studies  of  microspira  comma,  he  found  its  composi- 
tion to  be  as  follows  :  water  88.3  per  cent.,  albumin  7.6 
per  cent.,  ash  3.6  per  cent.  The  dry  substance  of  the 
bacteria  contains  the  following :  albumin  65  per  cent., 
ash  31  per  cent.  From  76  to  80  per  cent,  of  the  ash 
consists  of  sodium  chloride  and  phosphate. 

In  size  the  bacteria  are  certainly  the  smallest  living 

1  E,  Cramer:  Avchiv  fur  Hygiene,  Bd.  xiii,,  xvi.,  xxii.,  and  xxviii. 


52  BACTERIOLOGY. 

creatures  with  which  we  have  acquaintance,  being  visible 
only  when  very  highly  magnified.  In  order  that  some 
conception  of  their  microscopic  dimensions  might  be 
formed,  it  has  been  computed  that  of  the  average  size 
bacteria  about  thirty  billion  would  be  required  to  weigh 
a  gramm^,  and  that  about  one  billion  seven  hundred 
million  of\he  small  spherical  forms  might  readily  be 
suspended  in  a  drop  of  water. 

THE    CLASSIFICATION    OF    BACTERIA    IN    FAMILIES. 

The  classification  of  bacteria  into  families,  genera, 
and  species  has  been  a  subject  of  much  labor  and  dis- 
cussion. It  is  impossible,  on  account  of  limited  knowl- 
edge of  certain  species,  to  make  the  classification  of  the 
bacteria  accurate. 

The  basis  for  modern  systems  of  classification  has 
been  the  most  pronounced  morphologic  characters.  The 
system  of  classification  which  has  proven  of  greatest 
value  is  that  proposed  by  Migula,  in  Engler  and  Prantl's 
Die  Natiirlichen  Pflanzenfamitien,  1896,  and  elaborated 
in  his  System  der  Bakterien,  1900.  The  nomenclature 
employed  in  the  description  of  the  bacteria  is  that  of 
Migula. 

SCHIZOMYCETES.  —  Bacteria.  —  Unicellular,  chloro- 
phyll-free organisms  which  reproduce  by  division  into 
one,  two,  or  three  directions  of  space.  Sexual  repro- 
duction is  absent.  Many  species  develop  endogenous 
spores.  Motility,  occurring  in  some  genera,  is  due  to  the 
presence  of  flagella ;  in  Beggiatoa  and  Spirochseta  the 
motile  organs  are  unknown. 
I.  ORDER:  EUBACTERIA. 

Cells  without  central  nuclei,  sulphur,  and  bacterial 
purpurin  ;  colorless  or  only  feebly  colored. 


CLASSIFICATION  OF  BACTERIA  IN  FAMILIES.    53 

1.  Family:   Coccacecc  (Zopf,  Migula). 

Cells  in  a  free  state,  spherical,  becoming  slightly 
elliptical  before  division.  Division  occurs  in 
one,  two,  or  three  directions  of  space.  Motile 
organs  are  present  only  in  a  few  species. 
Endospore  formation  not  known  to  occur. 

1.  Genus:  Streptococcus  (Billroth). — Cells  spher- 

ical and  without  motile  organs.  Division 
only  in  one  direction  of  space.  After  divi- 
sion the  cells  separate  or  they  remain  for 
a  shorter  or  longer  time  in  apposition  and 
frequently  form  long  chains.  Usually  two 
cells  are  seen  lying  close  together  with  a 
slightly  greater  interval  between  the  next 
two  members  in  the  chain. 

2.  Genus:  Micrococcus  (Hallier,   Cohn). — Cells 

which  in  their  free  state  are  spherical. 
Division  in  two  directions  of  space.  If  the 
cells  after  division  remain  for  a  shorter  or 
longer  time  in  apposition  they  form  simple 
or  flat  aggregations  of  the  cells  in  which  the 
opposing  sides  of  the  organisms  are  flat- 
tened. Motile  organs  are  absent.  Endo- 
spore formation  has  not  been  demonstrated. 

3.  Genus:  Sarcina   (Goodsir). — Cells  which   in 

their  free  state  are  spherical.  Division  in 
three  directions  of  space,  forming  the  well- 
known  packet-form  aggregations.  Besides 
this,  cells  frequently  occur  singly  and  as 
diplococci  or  tetracocci  or  in  irregular 
aggregations.  Motile  organs  are  absent. 
Endospore  formation  has  not  been  definitely 
demonstrated. 


54  BACTERIOLOGY. 

4.  Genus:  Planococcus  (Migula). — Single  cells, 

spherical,  hut  usually  showing  aggregations 
of  two  of  four  cells.  Division  in  two  direc- 
tions of  space.  Motile  organs  are  present 
in  the  form  of  one  or  two  long  wavy  fla- 
gella.  Endospore  formation  does  not  occur. 
Only  a  few  species  are  known  of  this  genus. 

5.  Genus:  Planosarcina  (Migula). — Single  cells, 

spherical,  division  in  three  directions  of 
space.  Usually  the  cells  remain  in  appo- 
sition as  diplococci  or  tetracocci,  less  fre- 
quently as  distinct  packet  forms.  Motile 
organs  are  present  in  the  form  of  shorter  or 
longer  flagella.  Endospore  formation  does 
not  occur.  Only  three  species  have  been 
described. 
2.  Family:  Bacteriacece. 

Cells  which  in  their  free  state  occur  as  cylin- 
drical rods  which  divide  only  in  one  direction 
of  space,   that  is,  at  right  angles  to  the  long 
axis  of  the  cylinder.  Cells  may  be  very  short, 
so  that  it  is  difficult  to  differentiate  them  from 
the  coccacese.   Division  occurs  in  some  species, 
as  far  as  observation   has  demonstrated,  not  in 
the  division  of  a  cell  into  two  daughter  cells, 
but  in  each   large  rod  a  number  of  cell  divi- 
sions in  various  stages  are  usually  seen. 
1.   Genus:    Bacterium    (Ehrenberg,    Migula).— 
shorter   or   longer   cylindrical    cells,  some- 
times threads  of  considerable  length  with- 
out flagella.     Endospore  formation  has  been 
demonstrated    in    a   number   of  species,  in 
others  it  is  absent. 


CLASSIFICATION  OF  BACTERIA   IN  FAMILIES.    55 

2.  Genus:  Bacillus  (Colin,  Migula). — Shorter  or 

longer  rod  forms,  sometimes  short  ovoid 
cells,  and  in  other  species  long  thread  forms. 
Motile,  with  flagella  distributed  over  the 
entire  body.  Endospore  formation  occurs 
in  many  species. 

3.  Genus:    Pseudomonas. — Shorter     or    longer 

cylindrical  cells,  sometimes  thread  forms, 
motile,  with  polar  flagella.  The  number  of 
flagella  varies  in  different  species  from  one 
to  ten.  Endospore  formation  is  present  in 
a  few  of  the  species. 
3.  Family  :  Spirallficece. 

Cells  more  or  less  curved,  at  times  forming  dis- 
tinct spirals  when  a  number  are  joined  end  to 
end.  Division  of  the  cells  in  one  direction  of 
space,  that  is,  at  right  angles  to  the  long  axis 
of  the  rod.  Endospore  formation  is  absent 
except  in  a  few  species.  Motility  is  usually 
present;  where  the  motile  organs  are  known 
they  are  polar. 

1.  Genus:  Spirosoma   (Migula). — Cells    usually 

twisted  in  rather  large  spirals,  non-motile, 
without  flagella,  stiff  without  flexibility. 
The  number  of  species  of  this  genus  is  very 
small. 

2.  Genus :  Microspira  (Schroter). — Cells  mostly 

comma-form  or  sausage  shaped,  curved  or 
joined  in  S-shaped  figures,  or  even  in  long 
spiral  chains.  Motile,  with  one  to  three 
polar  flagella.  Endospore  formation  has 
not  been  demonstrated. 

3.  Genus:  Spirillum  (Ehrenberg). — Twisted  rods 


56  BACTERIOLOGY. 

of  variable  thickness  and  length,  frequently 
forming  long  spirals.  Endospore  formation 
has  been  observed  in  a  few  species.  The 
cells  are  motile  and  possess  flagella  at  one 
or  both  poles. 

4.  Genus:  Spirochceta. — Cells  of  spiral  form, 
thin  but  usually  quite  long,  motile  and  flexi- 
ble, snake-like  but  also  screw-like  in  motion. 
Motile  organs  are  unknown.  Endospore 
formation  has  not  been  observed. 
4.  Family :  Chlamydobacteriacece. 

Cells  cylindrical  and  thread  forms,  surrounded 
with  a  sheath.  Multiplication  results  through 
motile  and  non-motile  gonidia  which  arise 
directly  from  vegetative  cells  and  develop  into 
threads. 

1.  Genus:     Chlamydothrix. — Cells     cylindrical, 

non-motile,  enclosed  in  a  sheath.  Fre- 
quently the  sheath  is  only  apparent  upon 
applying  reagents.  Multiplication  through 
non-motile  round  or  ovoid  gonidia  that  are 
derived  directly  from  the  vegetative  cells. 
Synonyms  :  Streptothrix  (Colin,  Migula)  ; 
Leptothnx  (Kiitzing). 

2.  Genus :     Crenothrix      (Cohn).— Thread-form 

bacteria  without  branching,  attached  at  one 
end,  showing  a  differentiation  of  base  and 
apex,  and  increasing  in  thickness  toward 
the  free  end.  Sheath  rather  thick.  The 
sheaths  of  old  threads  in  waters  containing 
iron  are  saturated  with  oxide  of  iron.  The 
cells  are  cylindrical,  sometimes  flattened. 
Multiplication  through  non-motile  gonidia, 


CLASSIFICATION  OF  BACTERIA   IN  FAMILIES.   f>7 

usually  round  in  form,  that  are  derived 
from  vegetative  cells  through  fission.  The 
cells  of  the  thicker  rods  divide  in  three 
directions  of  space,  those  of  the  thinner 
threads  only  at  right  angles  to  the  long  axis 
of  the  threads. 

3.  Genus:    Phragmidiothrix    (Engler). — Thread 

bacteria  with  very  delicate  barely  visible 
sheath,  sometimes  100  microns  long  and  3 
to  12  microns  wide.  Cells  cylindrical,  later 
flattened.  Multiplication  through  non- 
motile  gonidia,  which  are  derived  from 
vegetative  cells  through  division  in  three 
directions  of  space.  Probably  similar  to 
crenothrix. 

4.  Genus:  Sphcerotilus  (Kutzing). — Cells  cylin- 

drical,   enclosed    in    the    sheaths,    dichoto- 
mously  branched  threads,  without  differen- 
tiation  of  base  and   apex.     Multiplication 
through    gonidia,    which    swarm    from    the 
sheaths  and  attach  themselves  to  objects  and 
develop  into  new  threads.  The  gonidia  have 
a  bunch  of  flagella  attached  to  one  pole. 
The  present  tendency  is  to  simplify  this  morpholog- 
ical  classification,   and   to    distribute   the  bacteria   into 
three  great  groups,  with  their  subdivisions,  the   mem- 
bers of  each  group  being  identified  by  their  individual 
outline,  viz.,  that  of  a  sphere,  a  rod,  or  a  spiral.     To 
these  three  grand  divisions  are  given  the  names  cocci  or 
micrococci,  bacilli,  and  spirilla. 

MODE  OF  MULTIPLICATION. — In 'the  group  micro- 
cocci  belong  all  spherical  forms — •/.  ^.,  all  those  forms 
the  isolated  individual  members  of  which  are  practically 


58 


BACTERIOLOGY, 


of  the  same  diameter  in  all  directions.     (See  Fig.  2,  u, 
b,  c,  d,  e.) 

FIG.  2. 

r\  _  Q  * 

°'oo 

c 


flO.o  0"    °^  •  °     °#  OU  ^-* 

<feV     &°       rf>  05,   *                    rri 

c*  °o%   o°0  ego                       tT 

c  d                                  e 

a.  Staphylococci.    6.  Streptococci,  c.  Diplococci.  d.  Tetrads,     e.  Sarcinse. 

FIG.  3. 


d  e  f 

a.  Bacilli  in  pairs.    6.  Single  bacilli,    cand  d.  Bacilli  in  threads. 

e  and  /.  Bacilli  of  variable  morphology. 

FIG.  4, 


^  -  '  <YI 

a  b  c  <i 

a  and  d.  Spirilla  in  short  segments  and  longer  threads  —the  so-called  comma 
forms  and  spirals,  b.  The  forms  known  as  spirochseta.  c.  The  thick  spirals 
sometimes  known  as  vibrios. 


CLASSIFICATION  OF  BACTERIA    IN  FAMILIES.    59 

The  bacilli  comprise  all  oval  or  rod-formed  bacteria. 
(See  Fig.  3.) 

To  the  spirilla  belong  all  organisms  that  are  curved 
when  seen  in  short  segments,  or  when  in  longer  threads 
are  twisted  in  the  form  of  a  corkscrew.  (See  Fig.  4.) 

The  micrococci  are  subdivided  according  to  their  pre- 
vailing mode  of  grouping,  as  seen  in  growing  cultures, 
into  staphylococci — those  growing  in  masses  like  clusters 
of  grapes  (see  Fig.  2,  a) ;  streptococci — those  growing  in 
chains  consisting  of  a  number  of  individuals  strung 
together  like  beads  upon  a  string  (see  Fig.  2,  6) ;  diplo- 
cocci — those  growing  in  pairs  (Fig.  2,  c)  ;  tetrads — those 
developing  as  fours  (Fig.  2,  d) ;  and  sarcince — those 
dividing  into  fours,  eights,  etc.,  as  cubes — that  is,  in 
contradistinction  to  all  other  forms,  the  segmentation, 
which  is  rarely  complete,  takes  place  regularly  in  three 
directions  of  space,  so  that  when  growing  the  bundle  of 
segmenting  cells  presents  somewhat  the  appearance  of  a 
bale  of  cotton  (Fig.  2,  e). 

To  the  bacilli  belong  all  straight,  rod-shaped  bacteria 
— *.  e.,  those  in  which  one  diameter  is  always  greater 
than  the  other. 

In  this  group  are  found  those  organisms  the  life- 
cycle  of  many  of  which  presents  deviations  from  the 
simple  rod-shape.  Many  of  them  in  the  course  of 
development  increase  in  length  into  long  threads, 
along  which  traces  of  segmentation  may  usually  be 
found — the  anthrax  bacillus  and  bacillus  cereus  are 
conspicuous  examples  of  this.  Again,  under  certain 
conditions,  many  of  them  possess  the  property  of  form- 
ing within  the  body  of  the  rods  oval,  glistening  spores 
(see  Fig.  6),  and,  if  the  conditions  are  not  altered,  the 
rods  may  entirely  disappear  and  nothing  be  left  in 


GO  BACTERIOLOGY. 

the  culture  but  these  oval  spores.  In  some  of  them 
this  phenomenon  of  spore-formation  is  accompanied  by 
an  enlargement  or  swelling  of  the  bacillus  at  the  point 
at  which  the  spore  is  located  (see  Fig.  6,  e  and  d). 
Again,  many  of  them,  from  unfavorable  conditions  of 
nutrition,  aeration,  or  temperature,  undergo  pathological 
changes — that  is,  the  individuals  themselves  experience 
degeneration  of  their  protoplasm  with  coincident  dis- 
tortion of  their  outline ;  they  are  then  usually  referred 
to  as  "involution-forms"  (see  Fig.  5,  a  and  b).  In 

FIG.  5. 

n , 


a.  Spirillum  of  Asiatic  cholera  (comma  bacillus) ;  normal  appearance 
in  fresh  cultures,  b.  Involution-forms  of  this  organism  as  seen  in  old 
cultures. 


all  of  these  conditions,  however,  so  long  as  death  has 
not  occurred,  it  is  possible  to  cause  these  forms  to  revert 
to  the  typical  rods  from  which  they  originated,  by  the 
renewal  of  conditions  favorable  to  their  normal  vege- 
tation. 

It  must  be  borne  in  mind,  though,  that  it  is  never 
possible  by  any  means  to  bring  about  changes  in  these 
organisms  that  will  result  in  the  permanent  conversion 
of  the  morphology  of  the  members  of  one  group  into 
that  of  another — that  is,  one  can  never  produce  bacilli 
from  micrococci,  nor  vice  versa;  and  any  evidence  which 
may  be  presented  to  the  contrary  is  based  upon  untrust- 
worthy methods  of  experimentation. 

Very  short  oval  bacilli  may  sometimes  be  mistaken 


GROUPING.  61 

for  micrococci,  and  at  times  micrococci  in  the  stage  of 
segmentation  into  diplococci  may  be  mistaken  for  short 
bacilli ;  but  by  careful  inspection  it  will  always  be 
possible  to  detect  a  continuous  outline  along  the  sides 
of  the  former,  and  a  slight  transverse  indentation  or 
partition-formation  between  the  segments  of  the  latter. 
The  high  index  of  refraction  of  spores,  the  property 
which  gives  to  them  their  glistening  appearance,  will 
always  serve  to  distinguish  them  from  micrococci.  This 
difference  in  refraction  is  especially  noticeable  if  the  illu- 
mination of  the  microscope  be  reduced  to  the  smallest 
possible  bundle  of  light-rays.  The  spores,  moreover, 
take  up  staining-reagents  much  less  readily  than  do 
the  micrococci.  The  most  reliable  differential  points, 
however,  are  the  infallible  properties  possessed  by  the 
spores  of  developing  into  bacilli,  and  by  the  spherical 
organism  with  which  they  may  have  been  confounded 
of  always  producing  other  micrococci  of  the  same 
spherical  form. 

A  convenient  classification  of  bacilli  is  that  based 
upon  constant  characteristics  which  are  seen  to  ap- 
pear in  the  course  of  their  development  under  spe- 
cial conditions — certain  of  them  possessing  the  power 
of  forming  spores,  while  from  others  this  peculiarity 
is  absent. 

We  have  less  knowledge  of  the  life-history  of  the 
spiral  forms.  Efforts  toward  their  cultivation  under 
artificial  conditions  have  thus  far  been  successful  in 
only  a  comparatively  limited  number  of  cases.  Mor- 
phologically, they  are  thread-  or  rod-like  bodies  which 
are  twisted  into  the  form  of  spirals.  In  some  of  them 
the  turns  of  the  spiral  are  long,  in  others  quite  short. 


62  BACTERIOLOGY. 

In  some  the  threads  appear  rigid,  in  others  flexible. 
They  are  motile  and  multiply  apparently  by  the  simple 
process  of  fission.1  In  most  respects,  save  form  and  the 
property  of  producing  spores,  they  are  analogous  in 
their  mode  of  growth  to  the  bacilli. 

The  micrococci  multiply  by  simple  fission.  When 
development  is  in  progress  a  single  cell  will  be  seen  to 
elongate  slightly  in  one  of  its  diameters.  Over  the 
centre  of  the  long  axis  thus  formed  will  appear  a  slight 
indentation  in  the  outer  envelope  of  the  cell ;  this  inden- 
tation will  increase  in  extent  until  there  exist  eventually 
two  individuals  which  are  distinctly  spherical,  as  was 
the  parent  from  which  they  sprang,  or  they  will  remain 
together  for  a  time  as  diplococci ;  the  surfaces  now  in 
juxtaposition  are  flattened  against  one  another,  and  not 
infrequently  a  fine,  pale  dividing-line  may  be  seen 
between  the  two  cells.  (See  Fig.  2,  c  and  d.)  A  similar 
division  in  the  other  direction  will  now  result  in  the 
formation  of  groups  of  fours  as  tetrads. 

In  the  formation  of  staphylococci  such  division  occurs 
irregularly  in  all  directions,  resulting  in  the  production 
of  the  clusters  in  which  these  organisms  are  commonly 
seen.  (See  Fig.  2,  a.)  With  the  streptococci,  however, 
the  tendency  is  for  the  segmentation  to  continue  in  one 
direction  only,  resulting  in  the  production  of  long  chains 
of  4,  8,  and  12  individuals.  (See  Fig.  2,  b.) 

The  sarchiEe  divide  more  or  less  regularly  in  three 
directions  of  space ;  but  instead  of  becoming  separated 
the  one  from  the  other  as  single  cells,  the  tendency  is 
for  the  segmentation  to  be  incomplete,  the  cells  remain- 
ing together  in  masses.  The  indentations  upon  these 
masses  or  cubes,  which  indicate  the  point  of  incomplete 

1  Dividing  into  two  transversely. 


SPORE-FORMATION.  63 

fission,  give  to  the  bundles  of  cells  the  appearance  com- 
monly ascribed  to  them,  viz.,  that  of  a  bale  of  cotton  or 
a  packet  of  rags.  (See  Fig.  2,  e.) 

The  mode  of  multiplication  of  bacilli  is  similar  to 
that  of  the  micrococci — i.  e.,  a  dividing  cell  elongates 
slightly  in  the  direction  of  its  long. axis;  an  indenta- 
tion appears  about  midway  between  its  poles,  and 
this  becomes  deeper  and  deeper,  until  eventually  two 
daughter-cells  have  formed.  This  process  may  occur 
in  such  a  way  that  the  two  young  bacilli  adhere 
together  by  their  adjacent  ends  in  much  the  same  way 
that  sausages  are  seen  to  be  held  together  in  strings 
(Fig.  3,/),  or  the  segmentation  may  take  place  more 
at  right  angles  to  the  long  axis,  so  that  the  proximal 
ends  of  the  young  cells  are  flattened,  while  the  distal 
extremities  may  be  rounded  or  slightly  pointed  (Fig. 
3,  e).  The  segmentation  of  the  anthrax  bacillus,  with 
which  we  are  to  become  acquainted  later,  results,  when 
completed,  in  an  indentation  of  the  adjacent  extrem- 
ities of  the  young  segments,  so  that  by  the  aid  of 
high  magnifying  powers  these  surfaces  are  seen  to  be 
actually  concave.  Bacilli  never  divide  longitudinally. 

SPORE-FORMATION. — With  the  spore-forming  bacilli, 
under  favorable  conditions  of  nutrition  and  temperature, 
the  same  mode  of  segmentation  is  seen  to  occur  during 
vegetation  ;  but  as  soon  as  these  conditions  become 
altered  by  the  exhaustion  of  nourishment,  the  presence 
of  detrimental  substances,  unfavorable  temperatures,  etc., 
they  enter,  in  f  their  life-cycle,  the  stage  to  which  we 
have  referred  as  spore-formation.  This  is  the  process 
by  which  the  organisms  are  enabled  to  enter  a  state  in 
which  they  resist  deleterious  influences  to  a  much  higher 
degree  than  is  possible  for  them  when  in  the  growing  or 
vegetative  condition, 


64  BACTERIOLOGY. 

In  the  spore,  resting,  or  permanent  state,  as  it  is  vari- 
ously called,  no  evidence  of  life  whatever  is  given  by  the 
spores  ;  though  as  soon  as  the  conditions  which  favor 
their  germination  have  been  renewed  these  spores  de- 
velop again  into  the  same  kind  of  cells  as  those  from 
which  they  originated,  and  the  appearances  observed  in 
the  vegetative  or  growing  stage  of  their  history  are 
repeated. 

Multiplication  of  spores,  as  such,  does  not  occur  ;  they 


FIG.  6. 


\  v> 


a  bed 

a.  Bacillus  subtilis  with  spores.  6.  Bacillus  anthracis  with  spores,  c.  Clos- 
tridium  form  with  spores,    d.  Bacillus  of  tetanus  with  end  spores. 

possess  only  the  power  of  developing  into  individual 
rods  of  the  same  nature  as  those  from  which  they  were 
formed,  but  not  of  giving  rise  to  a  direct  reproduction  of 
spores. 

When  the  conditions  which  favor  spore-formation 
present,  the  protoplasm  of  the  vegetative  cells  is  seen 
to  undergo  a  change.  It  loses  its  normal  homogeneous 
appearance  and  becomes  marked  by  granular,  refractive 
points  of  irregular  shape  and  size.  These  eventually 
coalesce,  leaving  the  remainder  of  the  cell  clear  and 
transparent.  When  this  coalescence  of  highly  refrac- 
tive particles  is  complete  the  spore  is  perfected.  In 
appearance  the  spore  is  oval  or  round,  and  very  highly 
refractive — glistening.  It  is  easily  differentiated  from 
the  remainder  of  the  cell,  which  now  consists  only 


SPORE-FORM  A  TION.  65 

of  a  cell-membrane  and  a  transparent,  clear  space 
which  surrounds  the  spore.  Eventually  both  the  cell- 
membrane  and  its  fluid  contents  disappear,  leaving  the 
oval  spore  free ;  it  then  gives  the  impression  of  being 
surrounded  by  a  dark,  sharply  defined  border.  When 
thus  perfectly  developed,  the  spore  may  be  regarded  as 
analogous  to  the  seeds  of  higher  plants.  Like  the  seed, 
it  evinces  no  evidence  of  life  until  placed  under  condi- 
tions favorable  to  germination,  when  there  develops 
from  it  a  cell  identical  in  all  respects  with  that  from 
which  it  originated.  Its  tenacity  of  life,  as  in  the  case 
of  seeds,  is  almost  unlimited.  It  may  be  kept  in  a  dry 
state,  and  this  has  actually  been  done,  for  years  without 
losing  the  power  of  germination.  The  glistening,  en- 
veloping spore-membrane  is  not  of  uniform  thickness 
throughout,  and  in  consequence  when  germination  oc- 
curs the  growing  bacillus,  the  so-called  vegetative  form 
of  the  organism,  protrudes  through  the  thinnest  part 
of  the  spore-membrane — that  is,  through  the  point  of 
least  resistance.  This  may  be  either  the  end  or  the  side 
of  the  spore,  according  to  the  species  under  observation. 
In  certain  cases  such  a  protrusion  is  not  observed,  but 
in  its  place  the  spore  in  toto  appears  to  be  gradually 
absorbed  or  in  some  way  converted  directly  into  a 
vegetating  cell.  It  evinces  no  motion  other  than  the 
mechanical  tremor  common  to  all  insoluble  microscopic 
particles  suspended  in  fluids,  and  it  remains  quiescent 
until  there  appear  conditions  favorable  to  its  subsequent 
development.  Occasionally  the  membrane  of  the  vege- 
tative cell  in  which  the  spore  is  formed  does  not  disap- 
pear from  around  it,  and  the  spore  may  then  be  seen 
lying  in  a  very  delicate  tubular  envelope.  Now  and 
then,  remnants  of  the  envelope  may  be  noticed  ad- 

5 


66  BACTERIOLOGY. 

bering   to   a   spore   which    has   not  yet   become    com- 
pletely free. 

By  the  ordinary  methods  of  staining,  spores  do  not 
become  colored,  so  that  they  appear  in  the  stained 
cells  as  pale,  transparent,  oval  bodies,  surrounded  by 
the  remainder  of  the  cell,  which  has  taken  up  the  dye. 

A  single  cell  produces  but  one  spore.  This  may  be 
located  either  at  an  extremity  or  in  the  centre  of  the 
cell.  (Fig.  6.) 

Occasionally  spore-formation  is  accompanied  by  an 
enlargement  of  the  cell  at  the  point  at  which  the  proc- 
ess is  in  progress.  As  a  result,  the  outline  of  the  cell 
loses  its  regular  rod  shape  and  becomes  that  of  a  club, 
a  drum-stick,  or  a  lozenge,  depending  upon  whether 
the  location  of  the  spore  is  to  be  at  the  pole  or  in  the 
centre  of  the  cell.  (See  Fig.  6,  e  and  d.) 

MOTILITY. — In  addition  to  the  property  of  spore-for- 
mation there  is  another  striking  difference  between  vari- 
ous species  of  the  rod-shaped  organisms,  namely,  the  prop- 
erty of  motility,  by  which  some  of  them  are  distinguished. 
This  power  of  motion  is  due  to  very  delicate,  hair- 
like  appendages  or  flagella,  by  the  lashing  motions  of 
which  the  rods  possessing  them  are  propelled  through 
the  fluid.  In  some  cases  the  flagella  are  located  at 
but  one  end  of  a  bacillus,  either  singly  (monotrichie)  or 
in  a  tuft  (lophotrichic) ;  and  in  some  cases,  especially 
with  the  bacillus  of  typhoid  fever,  they  are  given  off 
from  the  whole  surface  of  the  rod  (peritrichic).  (See  Fig. 
7.)  In  a  few  instances  similar  locomotive  organs  have 
been  detected  on  spherical  bacteria—^.  e.y  motile  micro- 
cocci  have  been  observed. 

For  a  long  time  this  property  of  independent  motion 
could  only  be  assumed  to  be  due  to  the  possession  of 


THERMAL  DEATH-POINT  OF  BACTERIA. 


67 


some  such  form  of  locomotive  apparatus,  because  similar 
appendages  had  been  seen  upon  some  of  the  large  motile 
spirilla  found  in  stagnant  water,  but  it  was  not  until  a 
few  years  ago  that  the  accuracy  of  this  assumption  was 
actually  demonstrated.  By  a  special  method  of  staining 
Loffler1  was  the  first  to  render  visible  these  hair-like 
appendages.  His  method,  as  well  as  the  several  modi- 
fications that  have  been  made  of  it,  depends  for  success 
upon  the  use  of  mordants,  through  the  agency  of  which 
the  stains  employed  are  caused  to  adhere  with  increased 
tenacity  to  the  objects  under  treatment. 


FIG.  7, 


a  b  c 

a.  Spiral  forms  with  a  flagellum  at  only  one  end.  6.  Bacillus  of  typhoid 
fever  with  flagella  given  off  from  all  sides,  c.  Large  spirals  from  stagnant 
water  with  wisps  of  flagella  at  their  ends  (spirillum  undula). 


THEEMAL  DEATH-POINT  OF  BACTERIA. — By  "  ther- 
mal death-point  of  bacteria  "  is  meant  the  temperature 
necessary  to  kill  them  in  a  given  time.  As  this  varies 
with  different  species,  it  is  an  aid  to  identification.  For 
the  practical  purposes  of  the  sanitarian  the  knowledge  is 
of  fundamental  importance.  The  thermal  death-point  of 
an  organism  is  ascertained  by  subjecting  it  to  varying  de- 
grees of  temperature  for  five  or  ten  minutes  until  the  point 

1  Loffler's  method  of  staining  will  be  found  in  the  chapter  devoted 
to  this  part  of  the  technique. 


68  BACTERIOLOGY. 

is  reached  where  it  is  killed.  The  test  is  best  carried 
out  by  means  of  small  glass  bulbs,  the  so-called  Stern- 
berg  bulbs,  or  through  the  use  of  capillary  tubes  con- 
taining a  small  amount  of  fluid  inoculated  with  the 
organism  to  be  studied.  The  bulb,  or  tube,  is  sealed  in 
the  gas  flame  and  placed  in  a  water-bath  kept  at  50°  C. 
for  five  minutes.  Sub-cultures  are  now  made  to  learn 
whether  the  bacteria  have  been  killed  or  not.  If  the  organ- 
ism survives  the  test  is  repeated  at  55°,  60°,  65°,  and 
70°  C.  Finally,  the  test  is  repeated  for  each  degree  of 
temperature  between  the  points  where  growth  is  still 
apparent  and  where  the  organisms  have  been  killed. 
If  the  bacteria  were  killed  when  heated  to  60°  C.  for 
five  minutes,  but  survived  when  heated  to  55°  C.,  then 
similar  tests  are  made  for  the  same  length  of  time  for 
each  degree  of  temperature  between  55°  and  60°  C.  It 
will  usually  be  found  that  heating  for  ten  minutes  suf- 
fices to  kill  the  bacteria  at  a  temperature  one  or  two  de- 
grees lower  than  that  required  when  heated  for  only  five 
minutes.  All  such  tests  should  be  made  at  least  in 
duplicate,  and  the  mean  of  the  results  taken. 


CHAPTER    III. 

Principles  of  sterilization  by  heat — Methods  employed — Discontinued 
sterilization — Fractional  sterilization — Apparatus  employed — Ster- 
ilization under  pressure — Sterilization  by  hot  air — Chemical  disin- 
fection and  sterilization — Mode  of  action  of  disinfectants — Practical 
disinfection. 

OF  fundamental  importance  to  successful  bacterio- 
logical manipulations  are  acquaintance  with  the  prin- 
ciples underlying  the  methods  of  sterilization  and  dis- 
infection, and  familiarity  with  the  approved  methods 
of  applying  these  principles  in  practice. 

In  many  laboratories  it  is  customary  to  employ  the 
term  sterilization  for  the  destruction  of  bacteria  by  heat, 
and  the  term  disinfection  for  the  accomplishment  of  the 
same  end  through  the  use  of  chemical  agents.  This 
distinction  in  the  use  of  the  terms  is  not  strictly  correct, 
as  we  shall  endeavor  to  explain. 

The  laboratory  application  of  the  word  sterilization 
for  the  destruction  of  bacteria  by  high  temperatures 
probably  arose  from  the  circumstance  that  culture- 
media,  and  certain  other  articles  that  it  is  desirable 
to  render  free  from  bacterial  life,  are  not  treated  by 
chemical  agents  for  this  purpose,  but  are  exposed  to 
the  influence  of  heat  in  various  forms  of  apparatus 
known  as  sterilizers ;  and  the  process  is,  therefore, 
known  as  sterilization.  On  the  other  hand,  cultures 
no  longer  useful,  bits  of  infected  tissue,  and  apparatus 
generally  that  it  is  desirable  to  render  free  from  danger, 
are  commonly  subjected  for  a  time  to  the  action  of  chem- 
ical compounds  possessing  germicidal  properties — i.  e., 

C'J 


70  BACTERIOLOGY. 

to  the  action  of  disinfectants ;  and  the  process  is,  there- 
fore, known  as  disinfection,  though  the  same  end  can 
also  be  reached  by  the  application  of  heat  to  these  arti- 
cles. Strictly  speaking,  sterilization  implies  the  com- 
plete destruction  of  the  vitality  of  all  micro-organisms 
that  may  be  present  in  or  upon  the  substance  to  be 
sterilized,  and  can  be  accomplished  by  the  proper  appli- 
cation of  both  thermal  and  chemical  agents ;  while 
disinfection,  though  it  may  insure  the  destruction  of  all 
living  forms  that  are  present,  need  not  of  necessity  do 
so,  but  may  be  limited  in  its  action  to  those  only  that 
possess  the  power  of  infecting  ;  it  may  or  may  not,  there- 
fore, be  complete  in  the  sense  of  sterilization.  From  this 
we  see  it  is  possible  to  accomplish  both  sterilization  and 
disinfection  as  well  by  chemical  as  by  thermal  means. 

In  practice  the  employment  of  these  means  is  gov- 
erned by  circumstances.  In  the  laboratory  it  is  essen- 
tial that  all  culture-media  with  which  work  is  to  be 
conducted  should  be  free  from  living  bacteria  or  their 
spores — they  must  be  sterile ;  and  it  is  equally  impor- 
tant that  their  original  chemical  composition  should 
remain  unchanged.  Tt  is  evident,  therefore,  that  ster- 
ilizatibn  of  these  substances  by  means  of  chemicals  is 
out  of  the  question,  for,  while  the  media  could  be  thus 
sterilized  j  it  would  be  necessary,  in  order  to  accomplish 
this,  to  add  to  them  substances  capable  not  only  of  de- 
stroying all  micro-organisms  present,  but  whose  pres- 
ence would  at  the  same  time  prevent  the  growth  of 
bacteria  that  are  to  be  subsequently  cultivated  in  these 
media — that  is  to  say,  after  performing  their  sterilizing 
or  germicidal  function  the  chemical  disinfectants  would, 
by  their  further  presence,  exhibit  their  antiseptic  prop- 
erties and  thus  render  the  material  useless  as  a  culture- 


STERILIZATION  BY  HEAT.  71 

medium.  Exceptions  to  this  are  seen,  however,  in  the 
case  of  certain  volatile  substances  possessing  disinfect- 
ant powers — chloroform  and  ether,  for  instance ;  these 
bodies,  after  exhibiting  their  germicidal  activities, 
may  be  driven  off  by  gentle  heat,  leaving  the  media 
quite  suitable  for  purposes  of  cultivation.  They  are 
not,  however,  in  general  u,se  in  this  capacity. 

The  circumstances  under  which  chemical  sterilization 
or  disinfection  is  practised  in  the  laboratory  are,  ordi- 
narily, either  those  in  which  it  is  desirable  to  render 
materials  free  from  danger  that  are  not  aifected  by  the 
chemical  action  of  the  agents  used,  such  as  glass  appa- 
ratus, etc.,  or  where  destructive  changes  in  the  compo- 
sition of  the  substances  to  be  treated,  as  in  the  rase 
of  old  cultures,  infected  tissues,  pathological  exudates, 
faeces,  etc.,  are  a  matter  of  no  consequence.  On  the 
other  hand,  for  the  sterilization  of  all  materials  to  be 
used  as  culture-media  heat  only  is  employed.1 

The  two  processes  will  be  explained  in  this  chapter, 
beginning  with 


STERILIZATION    BY    HEAT. 

Sterilization  by  means  of  high  temperature  is  accom- 
plished in  several  ways,  viz.,  by  subjecting  the  articles 
to  be  treated  to  a  high  temperature  in  a  properly  con- 
structed  oven — this  is  known  as  dry  sterilization ;  by 
subjecting  them  to  the  action  of  streaming  or  live  steam 
at  the  temperature  of  100°  C. ;  and  by  subjecting  them 
to  the  action  of  steam  under  pressure,  under  which 

1  An  occasional  exception  to  this  is  the  use  of  chloroform,  mentioned 
above. 


72  BACTERIOLOGY. 

circumstance  the  temperature  to  which  they  are  ex- 
posed becomes  more  and  more  elevated  as  the  pressure 
increases. 

Experience  has  taught  us  that  the  process  of  ster- 
ilization by  dry  heat  is  of  limited  application  because 
of  its  many  disadvantages.  For  successful  sterilization 
by  the  method  of  dry  heat,  not  only  is  a  relatively 
high  temperature  needed,  but  the  substances  under 
treatment  must  be  exposed  to  this  temperature  for  a 
comparatively  long  time.  The  penetration  of  dry  heat 
into  materials  which  are  to  be  sterilized  is,  moreover, 
much  less  thorough  than  that  of  steam.  Many  sub- 
stances of  vegetable  and  animal  origin  are  rendered 
valueless  by  subjection  to  the  dry  method  of  sterilization. 
For  these  reasons  comparatively  few  materials  can  be 
sterilized  in  this  way  without  seriously  impairing  their 
further  usefulness. 

Successful  sterilization  by  dry  heat  cannot  usually 
be  accomplished  at  a  temperature  lower  than  150°  C., 
and  to  this  degree  of  heat  the  objects  should  be  sub- 
jected for  not  less  than  one  hour.  For  the  sterilization, 
therefore,  of  the  organic  materials  of  which  the  media 
employed  in  bacteriological  work  are  composed,  and  of 
domestic  articles,  such  as  cotton,  woollen,  wooden,  and 
leather  articles,  this  method  is  wholly  unsuitable.  In 
bacteriological  work  its  application  is  limited  to  the 
sterilization  of  glassware  principally — such,  for  example^ 
as  flasks,  plates,  small  dishes,  test-tubes,  pipettes — and 
such  metal  instruments  as  are  not  seriously  injured  by 
the  high  temperature. 

METHODS  EMPLOYED. — Sterilization  by  moist  heat 
— steam — offers  conditions  much  more  favorable.  The 


STERILIZATION  BY  HEAT.  73 

penetrating  power  of  the  steam  is  not  only  more  ener- 
getic, but  the  temperature  at  which  sterilization  is  ordi- 
narily accomplished  is,  as  a  rule,  not  destructive  to  the 
objects  under  treatment.  This  is  conspicuously  seen  in 
the  work  of  the  laboratory;  the  culture-media,  com- 
posed in  the  main  of  decomposable  organic  materials 
that  would  be  rendered  entirely  worthless  if  exposed  to 
the  dry  method  of  sterilization,  sustain  no  injury  what- 
ever when  intelligently  subjected  to  an  equally  effective 
sterilization  with  steam.  The  same  may  be  said  of  cot- 
ton and  woollen  fabrics,  bedding,  clothing,  etc. 

Aside  from  the  relations  of  the  two  methods  to  the 
materials  to  be  sterilized,  their  action  toward  the  or- 
ganisms to  be  destroyed  is  quite  different.  The  pene- 
trating power  of  steam  renders  it  by  far  the  more  effi- 
cient agent  of  the  two.  The  spores  of  several  organisms 
which  are  killed  by  an  exposure  of  but  a  few  moments 
to  the  action  of  steam,  resist  the  destructive  action  of 
dry  heat  at  a  higher  temperature  for  a  much  greater 
length  of  time. 

These  differences  will  be  strikingly  brought  out  in 
the  experimental  work  on  this  subject.  For  our  pur- 
poses it  is  necessary  to  remember  that  the  two  methods 
have  the  following  applications  : 

The  dry  method,  at  a  temperature  of  150°-180°  C. 
for  one  hour,  is  employed  for  the  sterilization  of  glass- 
ware, such  as  flasks,  test-tubes,  culture-dishes,  pipettes, 
plates,  etc. 

Sterilization  by  steam  is  practised  with  all  culture- 
media,  whether  fluid  or  solid.  Bouillon,  milk,  gelatin, 
agar-agar,  potato,  etc.,  are  under  no  circumstances  to 
be  subjected  to  dry  heat. 

DISCONTINUED    STERILIZATION. — The     manner    in 


74  BACTERIOLOGY. 

which  heat  is  employed  in  processes  of  sterilization 
varies  with  circumstances.  When  used  in  the  dry 
form  its  application  is  always  continuous — /.  c.f  the 
objects  to  be  sterilized  are  simply  exposed  to  the 
proper  temperature  for  the  length  of  time  necessary 
to  destroy  all  living  organisms  which  may  be  upon 
them.  With  the  use  of  steam,  on  the  other  hand,  the 
articles  to  be  sterilized  are  frequently  of  such  a  nature 
that  a  prolonged  application  of  heat  might  materially  in- 
jure them.  For  this  and  other  reasons  steam  is  usually 
applied  intermittently  and  for  short  periods  of  time.  The 
principles  involved  in  the  intermittent  method  of  sterili- 
zation depend  upon  differences  of  resistance  to  heat  which 
the  organisms  to  be  destroyed  are  known  to  possess  at 
different  stages  of  their  development.  During  the  life- 
cycle  of  many  of  the  bacilli  there  is  a  stage  in  which 
the  resistance  of  the  organism  to  the  action  of  both 
chemical  and  thermal  agents  is  much  greater  than  at 
other  stages  of  their  development.  This  increased 
power  of  resistance  appears  when  these  organisms  are 
in  the  spore-  or  resting-stage,  to  which  reference  has 
already  been  made.  When  in  the  vegetative  or  grow- 
ing stage  most  bacteria  are  killed  in  a  short  time  by  a 
relatively  low  temperature ;  whereas,  under  conditions 
which  favor  the  production  of  spores,  the  spores  are 
seen  to  be  capable  of  resisting  very  much  higher  tem- 
peratures for  an  appreciably  longer  time ;  indeed,  spores 
of  certain  bacilli  have  been  encountered  that  retain  the 
power  of  germinating  after  an  exposure  of  from  five  to 
six  hours  to  the  temperature  of  boiling  water.  Such 
powers  of  resistance  have  never  been  observed  in  the 
vegetative  stage  of  development.  These  differences  in 
resistance  to  heat  which  the  spore-forming  organisms 
possess  at  their  different  stages  of  development  are 


STERILIZATION  BY  HEAT.  75 

taken  advantage  of  in  the  process  of  sterilization  by 
steam  known  as  the  discontinuous,  fractional,  or  inter- 
mittent method,  and  are  the  essential  feature  of  the 
principles  on  which  the  method  is  based. 

As  culture-media  are  dependent  for  their  usefulness 
upon  the  presence  of  more  or  less  unstable  organic 
compounds,  the  object  aimed  at  in  this  method  is  to 
destroy  the  organisms  in  the  shortest  time  and  with 
the  least  amount  of  heat.  It  is  accomplished  by  sub- 
jecting them  to  the  elevated  temperature  at  a  time 
when  the  bacteria  are  in  the  vegetating  or  growing  stage 
— i.  e.,  the  stage  at  which  they  are  most  susceptible  to 
detrimental  influences.  In  order  to  accomplish  this  it  is 
necessary  that  there  should  exist  conditions  of  tempera- 
ture, nutrition,  and  moisture  which  favor  the  vegetation 
of  the  bacilli  and  the  germination  of  any  spores  that 
may  be  present.  When,  as  in  freshly  prepared  nutrient 
media,  this  combination  is  found,  the  spore-forming  or- 
ganisms are  not  only  less  likely  to  enter  the  spore-stage 
than  when  their  environments  are  less  favorable  to  their 
vegetation,  but  spores  which  may  already  exist  develop 
very  quickly  into  mature  cells. 

It  is  plain,  then,  that  with  the  first  application  of 
steam  to  the  substance  to  be  sterilized  the  mature  vege- 
tative forms  are  destroyed  ;  while  certain  spores  that 
may  be  present  resist  this  treatment,  providing  the 
sterilization  is  not  continued  for  too  long  a  time.  If 
now  the  sterilization  be  discontinued,  and  the  material 
which  presents  conditions  favorable  to  the  germination 
of  the  spores  be  allowed  to  stand  for  a  time,  usually 
for  about  twenty-four  hours,  at  a  temperature  of  from 
20°  to  22°  C.,  those  spores  which  resisted  the  action  of 
the  steam  will,  in  the  course  of  this  interval,  germinate 


76  BACTERIOLOGY. 

into  the  less  resistant  vegetative  cells.  A  second  short 
exposure  to  the  steam  kills  these  forms  in  turn,  and 
by  a  repetition  of  this  process  all  bacteria  that  were 
present  may  be  destroyed  without  the  application  of 
the  steam  having  been  of  long  duration  at  any  time. 
It  should  be  remembered  that  while  spores  which 
may  be  present  are  not  directly  killed  by  such  an 
exposure  to  heat  as  they  experience  in  the  intermit- 
tent method  of  sterilization,  still  their  power  of  ger- 
mination is  somewhat  inhibited  by  this  treatment.  In 
this  method,  therefore,  if  the  temperature  of  100°  C. 
be  employed  for  too  long  a  time,  it  is  possible  so  to 
retard  the  germination  of  the  spores  as  to  render  it 
impossible  for  them  to  develop  into  the  vegetative  stage 
during  the  interval  between  the  heatings.  By  exces- 
sively long  exposures  to  high  temperature,  but  not  long 
enough  to  destroy  the  spores  directly,  the  object  aimed 
at  in  the  method  may  be  defeated,  and  in  the  end  the 
substance  undergoing  sterilization  be  found  still  to  con- 
tain living  bacteria.  In  this  process  the  plan  that  has 
given  most  satisfactory  results  is  to  subject  the  materials 
to  be  sterilized  to  the  action  of  steam,  under  the  ordi- 
nary conditions  of  atmospheric  pressure,  for  fifteen  min- 
utes on  each  of  three  successive  days,  and  during  the 
intervals  to  maintain  them  at  a  temperature  of  about 
25°-30°  C.  At  the  end  of  this  time  all  living  organ- 
isms which  were  present  will,  as  a  general  rule,  have 
been  destroyed,  and,  unless  opportunity  is  given  for 
the  access  of  new  organisms  from  without,  the  sub- 
stances thus  treated  remain  sterile.  As  an  exception 
to  this,  certain  species  of  spore-forming  bacteria  are 
occasionally  encountered  that  are  not  readily  destroyed 
by  this  mode  of  treatment.  These  species  are  found  so 


STERILIZATION  BY  HEAT.  77 

uniformly  in  the  soil  that  the  customary  designation  for 
them  is  that  of  "  the  soil  bacteria."  This  group  includes 
a  number  of  species  that  are  endowed  with  remarkable 
resistance  to  heat.  Some  of  them  are  probably  ther- 
mophilic  by  nature,  which  would  account  not  only  for 
the  failure  to  destroy  their  spores  by  the  ordinary  ex- 
posures to  steam,  but  also  for  their  slow  and  incomplete 
development  from  the  spore  to  the  less  resistant  vege- 
tative stage  during  the  intervals  between  the  heatings, 
for,  as  a  rule,  the  materials  containing  them  are  kept  at 
a  temperature  during  these  intervals  that  is  too  low  to 
favor  the  rapid  germination  of  the  species  having  ther- 
mophilic  tendencies. 

As  a  result  of  the  presence  of  these  species,  media 
that  have  been  subjected  to  the  customary  discontin- 
uous method  of  sterilization  may,  after  having  been 
kept  for  a  time,  reveal  the  presence  of  isolated  col- 
onies of  bacteria  distributed  through  them  in  such  a 
way  as  to  preclude  all  likelihood  of  their  having  fallen 
upon  it  from  the  air  after  sterilization  was  supposedly 
complete. 

Theobald  Smith l  has  called  attention  to  an  instruc- 
tive personal  experience.  He  finds  that  when  media 
are  present  in  vessels  in  only  thin  layers  the  spores  of 
anaerobic  species  do  not  develop  into  the  vegetative 
forms  during  the  interval  between  the  heatings,  for  the 
reason  that  the  shallow  layer  of  medium  does  not  suf- 
ficiently exclude  free  oxygen  to  permit  it ;  and  by  sub- 
jecting such  materials,  apparently  sterilized  by  the  inter- 
mittent method,  to  strictly  anaerobic  conditions  a  devel- 
opment of  anaerobic  species  will  often  occur.  On  the 

1  Theobald  Smith:  Journal  of  Experimental  Medicine,  vol.  iii.  No. 
6,  p.  647. 


78  BACTERIOLOGY. 

other  hand,  if  the  vessels  be  nearly  filled  with  media, 
and  especially  if  the  area  of  the  surface  be  small,  the 
conditions  are  much  more  favorable  to  the  germination 
of  anaerobic  spores,  and  sterilization  by  this  process  after 
such  precautions  is  usually  perfect. 

Fortunately,  these  undesirable  experiences  are  rare, 
but  that  they  do  occur,  and  result  in  no  small  degree 
of  annoyance,  will  be  admitted  by  most  bacteriologists. 

It  must  be  borne  in  mind  that  this  method  of  steril- 
ization is  only  applicable  in  those  cases  which  present 
conditions  favorable  to  the  germination  of  the  spores 
into  mature  vegetative  cells.  Dry  substances,  such  as 
instruments,  bandages,  apparatus,  etc.,  or  organic  ma- 
terials in  which  decomposition  is  far  advanced,  where 
conditions  of  nutrition  favorable  to  the  germination  of 
spores  are  not  present,  do  not  offer  the  conditions  requi- 
site for  the  successful  operation  of  the  principles  under- 
lying the  intermittent  method  of  sterilization. 

FRACTIONAL  STERILIZATION. — The  process  of  frac- 
tional sterilization  at  low  temperatures  is  based  upon  ex- 
actly the  same  principle,  but  differs  in  two  respects  from 
the  foregoing  in  the  manner  by  which  it  is  practised,  viz., 
it  requires  a  greater  number  of  exposures  for  its  accom- 
plishment, and  the  temperature  at  which  it  is  conducted  is 
not  above  68°— 70°  C.  It  is  employed  for  the  sterilization 
of  easily  decomposable  materials,  which  would  be  rendered 
useless  by  steam,  but  which  are  unaltered  by  the  tem- 
perature employed,  and  for  certain  albuminous  culture- 
media  that  it  is  desirable  to  retain  in  a  fluid  condition 
during  sterilization,  but  which  would  be  coagulated  if 
exposed  to  higher  temperatures.  This  process  requires 
that  the  material  to  be  sterilized  should  be  subjected  to 


STERILIZATION  BY  HEAT.  79 

a  temperature  of  68°-70°  C.  for  one  hour  on  each  of 
six  successive  days,  the  interval  of  twenty-four  hours 
between  the  exposures  admitting  of  the  germination 
of  spores  into  mature  cells.  During  this  interval  the 
substances  under  treatment  are  kept  at  about  25°— 
30°  C.  The  temperature  employed  in  this  process 
suffices  to  destroy,  in  about  one  hour,  the  vitality  of 
almost  all  organisms  in  the  vegetative  stage.  Formerly 
blood-serum  was  always  sterilized  by  the  intermittent 
method  at  a  low  temperature. 

STERILIZATION  UNDER  PRESSURE.  —  Sterilization 
by  steam  is  also  practised  by  what  may  be  called 
the  direct  method — that  is  to  say,  both  the  mature 
organisms  and  the  spores  which  may  be  present  in 
the  material  to  be  sterilized  are  destroyed  by  a  single 
exposure  to  the  steam.  In  this  method  steam  at  its  ordi- 
nary temperature  and  pressure — live  steam  or  streaming 
steam,  as  it  is  called — is  employed  just  as  in  the  first 
method  described ;  but  it  is  allowed  to  act  for  a  much 
longer  time,  usually  for  not  less  than  an  hour ;  or  steam 
under  pressure,  and  consequently  of  a  higher  tempera- 
ture, is  now  frequently  employed.  By  the  latter  pro- 
cedure a  single  exposure  of  fifteen  minutes  is  sufficient 
for  the  destruction  of  practically  all  bacilli  and  their 
spores,  providing  the  pressure  of  the  steam  is  not  less 
than  one  atmosphere  over  and  above  that  of  normal ; 
this  is  approximately  equivalent  to  a  temperature  of 
122°  C.  to  which  the  organisms  are  exposed. 

The  objection  that  has  been  urged  to  both  of  these 
methods,  particularly  that  in  which  steam  under  press- 
ure is  employed,  is  that  the  properties  of  the  media 
are  altered.  Gelatin  is  said  to  become  cloudy  and  lose 
the  property  of  solidifying  ;  in  bouillon  and  agar-agar 


80  BACTERIOLOGY. 

fine  precipitates  are  said  to  result,  and  some  believe 
the  reaction  undergoes  a  change.  In  the  experience 
of  those  who  have  used  steam  under  pressure  not  ex- 
ceeding one  atmosphere  for  ten  to  fifteen  minutes  these 
obstacles  have  rarely  been  encountered.  There  is  one 
point  to  be  borne  in  mind,  however,  in  using  steam 
under  pressure,  viz.,  it  is  not  possible  to  regulate  the 
time  of  exposure  to  the  same  degree  of  nicety  as  where 
ordinary  live  steam  is  used.  The  reason  for  this  is 
that  if  the  apparatus  be  opened  to  remove  the  objects 
being  sterilized  while  the  steam  within  it  is  under 
pressure,  the  escape  of  steam  will  be  so  rapid  that  all 
fluids  within  the  chamber,  thus  suddenly  relieved  of 
pressure,  will  begin  to  boil  violently,  and,  as  a  rule, 
will  boil  quite  out  of  the  tubes,  flasks,  etc.,  containing 
them.  For  this  reason  the  apparatus  must  be  kept 
closed  until  cool,  or  until  the  gauge  indicates  that  press- 
ure no  longer  exists  within  the  chamber,  and  even  then 
it  should  be  opened  very  cautiously.  It  is  patent  from 
this  that  the  temperature  and  time  of  exposure  of  arti- 
cles sterilized  by  this  process  cannot  usually  be  con- 
trolled with  accuracy.  It  requires  some  time  to  reach  a 
given  pressure  after  the  apparatus  is  closed,  and  it  also 
requires  time  for  cooling  after  the  desired  exposure  to 
such  pressure  before  the  apparatus  can  be  opened. 

It  is  manifest  that  during  these  three  periods,  viz., 
(a)  reaching  the  pressure  desired,  (b)  time  during  which 
the  pressure  is  maintained,  and  (c)  time  for  fall  of  press- 
ure before  the  chamber  can  be  opened,  it  is  difficult  to 
say  certainly  to  what  temperature  and  pressure  the  arti- 
cles in  the  apparatus  have,  on  the  whole,  been  subjected. 
Clearly,  if  the  desired  pressure  and  temperature  have 
been  maintained  for  ten  minutes,  one  cannot  say  that 


STERILIZATION  BY  HEAT. 


81 


that  is  all  the  heat  to  which  the  articles  have  been  sub- 
jected during  their  stay  in  the  chamber.  In  this  light, 
while  steam  under  pressure  may  answer  very  well  for 
routine  sterilization,  still  it  presents  insurmountable 
obstacles  to  its  use  in  more  delicate  experiments  where 
time-exposure  to  definite  temperature  is  of  importance. 
Nevertheless,  for  general  laboratory  purposes,  sterili- 
zation by  steam  under  pressure  has  so  much  to  recom- 
mend it  in  the  way  of  economy  of  time  and  certainty 
of  accomplishment  that  it  has  practically  superseded 
the  older  methods  of  sterilization  by  streaming  or  live 


Steam  sterilizer,  pattern  of  Koch. 

steam ;  and  in  most  laboratories  the  original  styles  of 
steam  sterilizers  are  rapidly  giving  way  to  some  one  or 
another  of  the  modern  forms  of  autoclave. 


82  BACTERIOLOGY. 

For  sterilization  by  live  steam  the  apparatus  in  com- 
mon use  was  for  a  long  time  the  cylindrical  boiler  rec- 
ommended by  Koch.  (See  Fig.  8.)  Its  construction 
is  very  simple,  essentially  that  of  the  ordinary  domestic 
potato-steamer.  It  consists  of  a  copper  cylinder,  the 
lower  fifth,  approximately,  of  which  is  somewhat  larger 
in  circumference  than  the  remaining  four-fifths  and 
serves  as  a  reservoir  for  the  water  from  which  the 
steam  is  to  be  generated.  Covering  this  section  of  the 
cylinder  is  a  wire  rack  or  grating,  through  which  the 
steam  passes,  and  which  supports  the  articles  to  be 
sterilized.  Above  this,  comprising  the  remaining  four- 
fifths  of  the  cylinder,  is  the  chamber  for  the  reception 
of  the  materials  over  and  through  which  the  steam  is  to 
pass.  The  cylinder  is  closed  by  a  snugly  fitting  cover, 
through  which  are  usually  two  perforations,  into  which 
a  thermometer  and  a  manometer  may  be  inserted.  The 
whole  of  the  outer  surface  of  the  apparatus  is  encased  in 
a  non-conducting  mantle  of  asbestos  or  felt. 

The  water  is  heated  by  a  gas-flame  placed  in  an  en- 
closed chamber,  upon  which  the  apparatus  rests,  which 
serves  to  diminish  the  loss  of  heat  and  deflection  of  the 
flame  through  the  action  of  draughts.  The  apparatus 
is  simple  in  construction,  and  the  only  point  which  is 
to  be  observed  while  using  it  is  the  level  of  the  water 
in  the  reservoir.  On  the  reservoir  is  a  water-gauge 
which  indicates  at  all  times  the  amount  of  water  in  the 
apparatus.  The  amount  of  water  should  never  be  too  small 
to  be  indicated  by  the  gauge ;  otherwise  there  is  danger 
of  the  reservoir  becoming  dry  and  the  bottom  of  the  ap- 
paratus being  destroyed  by  the  direct  action  of  the  flame. 

A  sterilizer  that  has  come  into  very  general  use  in 
bacteriological  laboratories  is  one  originally  intended 


STERILIZATION   UNDER   PRESSURE. 


83 


for  use  in  the  kitchen.  It  is  called  the  "  Arnold  steam 
sterilizer."  It  is  very  ingenious  in  its  construction  as 
well  as  economical  in  its  employment. 

The  difference  between  this  apparatus  and  that  just 
described  is  that  it  provides  for  the  condensation  of  the 
steam  after  its  escape  from  the  sterilizing  chamber,  and 
returns  the  water  of  condensation  automatically  to  the 

FIG.  9. 


Arnold  steam  sterilizer. 

reservoir,  so  that  in  practice  the  apparatus  requires  but 
little  attention,  as  with  ordinary  care  there  is  no  likeli- 
hood of  the  water  in  the  reservoir  becoming  exhausted, 
with  the  consequent  destruction  of  the  sterilizer. 
Fig.  9  shows  a  section  through  this  apparatus. 


STERILIZATION   UNDER   PRESSURE. 

The  advantages  of  the  use  of  steam  under  pressure 
for  the   purposes   of  sterilization   have  received  such 


84 


BACTERIOLOGY. 


general  recognition  that  almost  everywhere  this  method 
is  supplanting  the  older  one  of  intermittent  sterilization 
with  streaming  or  live  steam.  By  this  plan  one  is  able 
to  accomplish,  by  a  single  exposure  of  fifteen  minutes  to 
steam  under  a  pressure  of  one  atmosphere,  the  same  end 
that  would,  with  streaming  .-steam,  require  three  expos- 
ures of  fifteen  minutes  on  each  of  three  successive  days. 

FIG.  10. 


Autoclave,  pattern  of  Wiesnegg.    A.  External  appearance.    B.  Section. 

For  sterilization  by  steam  under  pressure  several  spe- 
cial forms  of  apparatus  exist.  The  principles  involved 
in  them  all  are,  however,  the  same.  They  provide  for<| 
the  generation  of  steam  in  a  chamber  from  which  it 
cannot  escape  when  the  apparatus  is  closed.  Upon  the 
cover  of  this  chamber  is  a  safety-valve,  which  can  be 
regulated  so  that  any  degree  of  pressure  (and  coinci- 


STERILIZATION  BY  HOT  AIR.  85 

dently  of  temperature)  that  is  desirable  may  be  main- 
tained within  the  sterilizing  chamber.  These  sterilizers 
are  known  as  "  digesters  "  and  as  "  autoclaves."  Their 


FIG.  11. 


Autoclave  or  digester  for  sterilizing  by  steam  under  pressure. 

construction    can  best  be  understood   by  reference  to 
Figs.  10  and  11. 

STERILIZATION    BY   HOT   AIR. 

The  hot-air  sterilizers  used  in  laboratories  are  sim- 
ply double-walled  boxes  of  Russian   or  Swedish  iron 


86 


BACTERIOLOGY. 


(Fig.  12),  having  a  double-walled  door,  which  closes 
tightly,  and  a  heavy  copper  bottom.  They  are  pro- 
vided with  openings  for  the  escape  of  the  contained  air 
and  the  entrance  of  the  heated  air.  The  flame,  usually 
from  a  rose-burner  (Fig.  13),  is  applied  directly  to  the 
bottom.  The  heat  circulates  from  the  lower  surface 
around  about  the  apparatus  through  the  space  between 
its  walls. 

FIG.  12. 


FIG.  13. 


Laboratory  hot-air  sterilizer. 


Rose-burner. 


The  construction  of  the  copper  bottom  of  the  appa- 
ratus upon  which  the  flame  impinges  is  designed  to  pre- 
vent the  direct  action  of  the  flame  upon  the  sheet-iron 
bottom  of  the  chamber.  It  consists  of  several  copper 
plates  placed  one  above  the  other,  but  with  a  space  of 
about  4  to  5  mm.  between  the  plates.  These  copper 
bottoms  after  a  time  become  burned  out,  and  unless 


CHEMICAL  STERILIZATION  AND  DISINFECTION.    87 

they  are  replaced  the  apparatus  is  useless.  The  older 
forms  of  hot-air  sterilizers  are  so  constructed  that  their 
repair  is  a  matter  involving  some  time  and  expense. 
To  meet  this  objection  I  had  constructed  some  years  ago 
a  sterilizer  in  all  respects  similar  to  the  old  form  except 
in  the  arrangement  of  the  copper  bottom.  This  latter 
is  made  in  such  a  way  that  it  can  easily  be  removed,  so 
that  by  keeping  several  sets  of  copper  plates  on  hand  a 
new  plate  can  readily  be  inserted  when  the  old  one  is 
burned  out. 

In  the  employment  of  the  hot-air  sterilizer  care 
should  always  be  given  to  the  condition  of  the  copper 
bottom ;  for  the  direct  application  of  heat  to  the  sheet- 
iron  plate  upon  which  the  substances  to  be  steril- 
ized stand  results  not  only  in  destruction  of  the  appa- 
ratus, but  frequently  in  destruction  of  the  substances 
undergoing  sterilization. 

Since  the  temperature  at  which  this  form  of  steril- 
ization is  usually  accomplished  is  high,  from  150°  to 
180°  C.,  it  is  well  to  have  the  apparatus  encased  in 
asbestos  boards,  to  diminish  the  radiation  of  heat  from 
its  surfaces.  This  not  only  confines  the  heat  to  the 
apparatus,  but  guards  against  the  destructive  action  of 
the  radiated  heat  on  woodwork,  furniture,  etc.,  that 
may  be  in  the  neighborhood. 

CHEMICAL   STERILIZATION   AND    DISINFECTION. 

As  has  been  stated,  it  is  possible  by  means  of  cer- 
tain chemical  substances  to  destroy  all  bacteria  and 
their  spores  that  may  be  within  or  upon  various  mate- 
rials and  objects — i.  e.,  to  sterilize  them ;  and  it  is  also 
possible  by  the  same  means  to  rob  objects  of  their 


88  BACTERIOLOGY. 

dangerous  infective  properties  without  at  the  same 
time  sterilizing  them — i.  e.,  to  disinfect  them.  This 
latter  process  depends  upon  the  fact  that  the  vital- 
ity of  many  of  the  less  resistant  pathogenic  organ- 
isms is  easily  destroyed  by  an  exposure  to  particular 
chemical  substances  that  may  be  without  effect  upon 
the  more  resistant  saprophytes  and  their  spores  that  are 
present. 

In  general,  the  use  of  chemicals  for  sterilization  is 
not  to  be  considered  in  connection  with  substances  that 
are  to  be  employed  as  culture-media,  and  their  employ- 
ment is  restricted  in  the  laboratory  to  materials  that 
are  of  no  further  value,  and  to  infected  articles  that  are 
not  injured  by  the  action  of  the  agents  used,  though  ex- 
ceptionally such  volatile  germicides  as  chloroform  and 
ether  are  employed  for  the  sterilization  of  special  culture- 
media.  (See  Preservation  of  Blood-serum  with  Chloro- 
form.) In  short,  they  are  mainly  of  value  in  rendering 
infected  waste -materials  innocuous.  For  the  successful 
performance  of  this  form  of  disinfection  there  is  one 
fundamental  rule  always  to  be  borne  in  mind,  viz.,  it 
is  essential  to  success  that  the  disinfectant  used  should 
come  in  direct  contact  with  the  bacteria  to  be  destroyed, 
otherwise  there  is  no  disinfection. 

For  this  reason  one  should  always  remember,  in 
selecting  the  disinfecting  agent,  the  nature  of  the  mate- 
rials containing  the  bacteria  upon  which  it  is  to  act,  for 
the  majority  of  disinfectants,  and  particularly  those  of 
an  inorganic  nature,  vary  in  the  degree  of  their  potency 
with  the  chemical  nature  of  the  mass  to  which  they  are 
applied.  Often  the  materials  containing  the  bacteria  to 
be  destroyed  are  of  such  a  character  that  they  combine 
with  the  disinfecting  agent  to  form  insoluble,  more  or 


CHEMICAL  STERILIZATION  AND  DISINFECTION.  80 

less  inert  precipitates ;  these  so  interfere  with  the  pene- 
tration of  the  disinfectant  that  many  bacteria  may  escape 
its  destructive  action  entirely  and  no  disinfection  be  ac- 
complished, although  an  agent  may  have  been  employed 
that  would,  under  other  circumstances,  have  given  en- 
tirely satisfactory  results. 

An  antiseptic  is  a  body  which,  by  its  presence,  pre- 
vents the  growth  of  bacteria  without  of  necessity  killing 
them.  A  body  may  be  an  antiseptic  without  possessing 
disinfecting  properties  to  any  very  high  degree,  but  a 
disinfectant  is  always  an  antiseptic  as  well. 

A  germicide  is  a  body  possessing  the  property  of 
killing  bacteria. 

MODE  OF  ACTION  OF  DISINFECTANTS. — In  the  de- 
struction of  bacteria  by  means  of  chemical  substances 
there  occurs,  most  probably,  a  definite  chemical  reac- 
tion— that  is  to  say,  the  characteristics  both  of  the 
bacteria  and  the  agent  employed  in  their  destruction 
are  lost  in  the  production  of  an  inert  third  body,  the 
result  of  their  combination.  It  is  impossible  to  state 
with  certainty,  as  yet,  that  this  is  in  general  the  case ;  but 
the  evidence  that  is  rapidly  accruing  from  studies  upon 
disinfectants  and  their  mode  of  action  points  strongly 
to  the  accuracy  of  this  belief.  This  reaction,  in  which 
the  typical  structures  of  both  bodies  concerned  are  lost, 
takes  place  between  the  agent  employed  for  disinfection 
and  the  protoplasm  of  the  bacteria.  For  example,  in 
the  reaction  that  is  seen  to  take  place  between  the  salts 
of  mercury  and  albuminous  bodies  there  results  a 
third  compound,  which  has  neither  all  the  character- 
istics of  mercury  nor  of  albumin,  but  partakes  of 
some  of  the  peculiarities  of  both ;  it  is  a  combina- 
tion of  albumin  and  mercury,  commonly  known  by  the 


90  BACTERIOLOGY. 

indefinite  term  "albuminate  of  mercury."  Some  such 
reaction  as  this  apparently  occurs  when  the  soluble  salts 
of  mercury  are  brought  in  contact  with  bacteria.  This 
view  has  been  strengthened  by  the  experiments  of 
Geppert,  in  which  the  reaction  was  caused  to  take  place 
between  the  spores  of  the  anthrax  bacillus  and  a  solu- 
tion of  mercuric  chloride,  the  result  being  the  apparent 
destruction  of  the  vitality  of  the  spores  by  the  forma- 
tion of  this  third  compound.  In  these  experiments  it 
was  shown  that  though  this  combination  had  taken 
place,  still  it  did  not  of  necessity  imply  the  death  of 
the  spores,  for  if  by  proper  means  the  combination  of 
mercury  with  their  protoplasm  was  broken  up,  many 
of  the  spores  resumed  their  vitality,  with  all  their  pre- 
vious disease-producing  and  cultural  peculiarities.  Gep- 
pert  employed  a  solution  of  ammonium  sulphide  for 
the  purpose  of  destroying  the  combination  of  spore- 
protoplasm  and  mercury ;  the  mercury  was  precipi- 
tated from  the  protoplasm  as  an  insoluble  sulphide, 
and  the  protoplasm  of  the  spores  returned  to  its  original 
condition.  These  and  other  somewhat  similar  experi- 
ments have  given  a  new  impulse  to  the  study  of  disin- 
fectants, and  in  the  light  shed  by  them  many  of  our 
previously  formed  ideas  concerning  the  action  of  disin- 
fecting agents  have  been  modified. 

The  process  of  disinfection  is  not  a  catalytic  one — 
i.  e.,  occurring  simply  as  a  result  of  the  presence  of  the 
disinfecting  body,  which  is  not  itself  decomposed  during 
its  process  of  destruction — but  is,  as  said,  a  definite  chem- 
ical reaction  occurring  within  more  or  less  fixed  limits ; 
that  is  to  say,  with  a  given  amount  of  the  disinfect- 
ant employed  so  much  work,  expressed  in  terms  of  disin- 
fection— destruction  of  bacteria — can  be  accomplished. 


CHEMICAL  STERILIZATION  AND  DISINFECTION.   91 

Another  point  in  favor  of  this  view  is  the  increased 
energy  of  the  reaction  with  elevation  of  temperature. 
Just  as  in  many  other  chemical  phenomena  the  inten- 
sity and  rapidity  of  the  reaction  become  greater  under 
the  influence  of  heat,  so  in  the  process  of  disinfection 
the  combination  between  the  disinfectant  and  the  organ- 
isms to  be  destroyed  is  much  more  energetic  at  a  tem- 
perature of  37°  to  39°  C.  than  it  is  at  12°  to  15°  C. 

A  number  of  important  and  novel  suggestions  with 
regard  to  the  modus  operandi  of  disinfection  were 
brought  out  through  the  work  of  Kronig  and  Paul,1 
who  took  up  the  subject  from  its  physico-chemical 
standpoint.  The  comprehensive  nature  of  this  elab- 
orate investigation  precludes  more  than  a  brief  men- 
tion of  some  of  the  conclusions  reached,  and  in  order 
that  these  may  be  intelligible,  certain  beliefs  (working 
hypotheses)  of  the  physical  chemists  should  be  borne  in 
mind.  In  1887  Arrhenius  proposed  the  theory  that 
when  an  electrolyte  (a  compound  decomposable  by  an 
electric  current)  is  dissolved  in  water  its  molecules  break 
down,  not  simply  into  their  component  atoms,  but  into 
ions,  which  are  atoms  or  groups  of  atoms  having  electro- 
positive and  electro-negative  characteristics.  According 
to  this  theory,  salts,  when  dissolved  in  water,  undergo 
electrolytic  dissociation  into  metallic  and  acidic  ions, 
the  former  being  the  electro-positive  cation,  the  latter 
the  electro-negative  anion;  sodium  chloride,  for  exam- 
ple, resolving  itself,  under  these  conditions,  into  its 
sodium,  or  metal-ion,  and  its  chlorine,  or  acidic  ion. 
The  electro-positive  cations,  according  to  Ostwald,  com- 
prise the  metals  and  metal -like  radicals,  such  as  am- 

1  Kriinig  and  Paul:  Zeitschrift  fur  Hygiene  und  Infectionskrunk- 
heiten,  1897,  vol.  xxv.  pp.  1-112. 


92  BACTERIOLOGY. 

moniurn  (NH4)  and  hydrogen  (H)  ;  while  the  electro- 
negative anions  include  the  halogens,  the  acidic  radicals 
(such  as  NO3  and  SO4),  and  hydroxyl.1  Using  this  theory 
as  the  basis  of  their  investigations,  Kronig  and  Paul 
reached  the  following  conclusions  with  regard  to  the 
action  of  chemical  disinfectants  : 

The  germicidal  value  of  a  metallic  salt  depends  not- 
only  upon  its  specific  character,  but  also  upon  that  of  its 
anion. 

Solutions  of  metallic  salts  in  which  the  metallic  part 
is  represented  by  a  complex  ion  and  in  which  the  con- 
centration of  the  metal  ion  is  very  slight,  have  but 
feeble  disinfecting  activity. 

The  halogen  compounds  of  mercury  act  according  to 
the  degree  of  their  dissociation. 

The  disinfecting  power  of  the  halogens — chlorine, 
bromine,  iodine — (as  well  as  their  compounds)  is  in  in- 
verse ratio  to  their  atomic  weights. 

The  disinfecting  activity  of  watery  solutions  of  mer- 
curic chloride  is  diminished  by  the  addition  to  them 
of  other  halogen  compounds  of  metals  and  of  hydro- 
chloric acid.  It  appears  probable  that  this  is  due  to 
obstruction  offered  to  electrolytic  dissociation. 

The  disinfecting  activities  of  watery  solutions  of  mer- 
curic nitrate,  mercuric  sulphate,  and  mercuric  acetate  are 
increased  by  the  moderate  addition  of  sodium  chloride. 

In  general,  acids  disinfect  according  to  the  degree 
of  their  dissociation — i.  e.y  according  to  the  concentra- 
tion of  their  hydrogen  ions  in  the  solution. 

1  Consult  Ostwald's  Lehrbuch  der  Allg.  Chemie  ;  or  Muir's  transla- 
tion of  Ostwald's  Solutions,  p.  189,  published  by  Longmans,  Green  & 
Co.,  London  and  New  York,  1891.  Also  "  The  Rise  of  the  Theory  of 
Electrolytic  Dissociation,"  etc.,  by  H.  C.  Jones,  Ph.  D.,  Johns  Hopkins 
Hospital  Bulletin,  No.  87,  June,  1898,  p.  136. 


CHEMICAL  STERILIZATION  AND  DISINFECTION.   93 

The  bases,  potassium,  sodium,  lithium,  and  ammo- 
nium hydroxide,  disinfect  according  to  the  degree  of 
their  dissociation — i.  e.,  corresponding  to  the  concen- 
tration of  their  hydroxyl  ions  in  the  solution. 

The  disinfecting  activity  of  metallic  salts  is,  in  gen- 
eral, less  in  albuminous  fluids  than  in  water.  It  is 
probable  that  this  is  due  to  a  diminution  in  the  concen- 
tration of  metallic  ions  in  the  solution. 

What  has  been  said  refers  more  particularly  to  the 
inorganic  salts  which  are  employed  for  this  purpose. 
It  is  probable  that  the  organic  bodies  possessing  dis- 
infectant properties  owe  this  power  to  some  such  similar 
reaction,  though,  as  yet,  these  substances  have  not  been 
so  thoroughly  studied  in  this  relation. 

The  reaction  between  the  inorganic  salts  and  albu- 
minous bodies  is  not  selective ;  they  combine  in  most 
instances  with  any  or  all  protoplasmic  bodies  present. 
For  this  reason  the  employment  of  many  of  the  com- 
moner disinfectants  in  general  practice  is  a  matter  of 
doubtful  advantage.  For  example,  the  disinfection  of 
excreta,  sputum,  or  blood,  containing  pathogenic  organ- 
isms, by  means  of  corrosive  sublimate,  is  a  procedure 
of  questionable  success.  The  amount  of  sublimate  em- 
ployed may  be  entirely  used  up  and  rendered  inactive 
as  a  disinfectant  by  the  ordinary  protoplasmic  sub- 
stances present,  without  having  any  appreciable  effect 
upon  the  bacteria  which  may  be  in  the  mass. 

These  remarks  are  introduced  in  order  to  guard 
against  the  implicit  confidence  so  often  placed  in  the 
disinfecting  value  of  corrosive  sublimate.  In  many 
bacteriological  laboratories  it  is  the  custom  to  keep  at 
hand  vessels  containing  solutions  of  corrosive  sublimate, 
into  which  infectious  materials  may  be  placed.  The 


94  BACTERIOLOGY. 

value  of  this  procedure,  as  we  have  just  learned,  may 
be  more  or  less  questionable,  especially  in  those  cases 
in  which  the  substance  to  be  disinfected  is  of  a  proteid 
nature  and  where  the  solution  used  is  not  freshly  pre- 
pared and  frequently  replenished.  On  the  introduction 
of  such  substances  into  the  sublimate  solution  the  mer- 
cury is  quickly  precipitated  by  the  albumin,  and  its  dis- 
infecting properties  may  be  in  large  part  or  entirely 
destroyed ;  we  may  in  a  very  short  time  have  little  else 
than  water  containing  an  inactive  precipitate  of  albumin 
and  mercury,  in  so  far  as  its  value  as  a  disinfectant  is 
concerned. 

Though  the  other  inorganic  salts  have  not  been  so 
thoroughly  studied  in  this  connection,  it  is  nevertheless 
probable  that  the  same  precautions  should  be  taken  in 
their  employment  as  we  now  know  to  be  necessary  in 
the  use  of  the  salts  of  mercury. 

PRACTICAL,  DISINFECTION. — Where  it  is  desirable 
to  use  chemical  disinfectants  in  the  laboratory,  much 
more  satisfactory  results  can  usually  be  obtained  from 
the  employment  of  carbolic  acid  in  solution.  A  3  or  4 
per  cent,  solution  of  commercial  carbolic  acid  in  water 
requires  longer  for  disinfection  ;  but  it  is,  at  the  same 
time,  open  to  fewer  objections  than  are  solutions  of  the 
inorganic  salts ;  though  here,  too,  we  find  a  somewhat 
analogous  reaction  between  the  carbolic  acid  and  proteid 
matters.  Under  ordinary  circumstances  its  action  is 
complete  in  from  twenty  minutes  to  one-half  hour.  It 
is  not  reliable  for  the  disinfection  of  resistant  spores ; 
such,  for  instance,  as  those  of  bacillus  anthrads. 

All  tissues  containing  infectious  organisms  should  be 
burned,  and  all  cloths,  test-tubes,  flasks,  and  dishes 
should  be  boiled  in  2  per  cent,  soda  (ordinary  washing- 


CHEMICAL  STERILIZATION  AND  DISINFECTION.    95 

soda)  solution  for  fifteen  to  twenty  minutes,  or  placed 
in  the  steam  sterilizer  for  half  an  hour. 

Intestinal  evacuations  may  best  be  disinfected  with 
boiling  water  or  with  milk  of  lime,  a  mixture  composed 
of  lime  in  solution  and  in  suspension — ordinary  fluid 
"  white-wash."  This  should  be  thoroughly  mixed  with 
the  evacuations  until  the  mass  contains  a  considerable 
excess  of  the  lime,  and  should  remain  in  contact  with 
them  for  one  or  two  hours.  Excreta  may  also  be  easily 
disinfected  by  thoroughly  mixing  them  with  two  or 
three  times  their  volume  of  boiling  water,  after  which 
they  are  kept  covered  until  cool. 

Sputum  in  which  tubercle  bacilli  are  present,  as  well 
as  the  vessel  containing  it,  must  be  boiled  in  2  per  cent, 
soda  solution  for  fifteen  minutes,  or  steamed  in  the  ster- 
ilizer for  at  least  half  an  hour. 

On  the  whole,  in  the  laboratory  we  should  rely  more 
upon  the  destructive  properties  of  heat  than  upon  those 
of  chemical  agents. 

From  what  has  been  said,  the  absurdity  of  sprink- 
ling here  and  there  a  little  carbolic  acid,  or  of  placing 
vessels  of  carbolic  acid  about  apartments  in  which  in- 
fectious diseases  are  in  progress,  must  be  plain.  Treat- 
ment of  water-closets  and  cesspools  by  allowing  now 
and  then  a  few  cubic  centimetres  of  some  so-called 
disinfectant  to  trickle  through  the  pipes  is  ridiculous. 
A  disinfectant  must  be  applied  to  the  bacteria,  and  must 
be  in  contact  with  them  for  a  long  enough  time  to  insure 
the  destruction  of  their  life. 

In  the  light  of  the  latest  experiments  upon  disin- 
fectants, the  place  formerly  occupied  by  many  agents 
in  the  list  of  substances  employed  for  the  purpose  will 
most  likely  be  changed  as  they  are  studied  more  closely. 


96  BACTERIOLOGY. 

The  agents,  then,  which  will  prove  of  greatest  value  in 
the  laboratory  for  the  purpose  of  rendering  infectious 
materials  harmless  are :  heat,  either  by  burning,  by 
steaming  for  from  half  an  hour  to  an  hour,  or  by  boil- 
ing in  a  2  per  cent,  sodium  carbonate  solution  for  fifteen 
minutes ;  3  to  4  per  cent,  solution  of  commercial  car- 
bolic acid ;  milk  of  lime,  and  a  solution  of  chlorinated 
lime  ("chloride  of  lime")  containing  not  less  than  0.25 
per  cent,  of  free  chlorine.  The  chloride  of  lime  from 
which  such  a  solution  is  to  be  made  should  be  fresh 
and  of  good  quality.  Good  chlorinated  lime,  as  pur- 
chased in  the  shops,  should  contain  not  less  than  25  to 
30  per  cent,  of  available  chlorine.  The  materials  to  be 
disinfected  in  either  of  the  lime  solutions  should  remain 
in  them  for  about  two  hours.  The  solutions  should  be 
freshly  prepared  when  needed,  as  they  rapidly  decom- 
pose upon  standing. 


CHAPTER   IV. 

Principles  involved  in  the  methods  of  isolation  of  bacteria  in  pure 
culture  by  the  plate  method  of  Koch— Materials  employed. 

As  was  stated  in  the  introductory  chapter,  the  isola- 
tion in  pure  cultures  of  the  different  species  that  may 
be  present  in  mixtures  of  bacteria  was  rendered  possible 
only  through  the  methods  suggested  by  Koch.  Since 
the  adoption  of  these  methods  they  have  undergone 
many  modifications,  but  the  fundamental  principle  re- 
mains the  same.  The  observation  which  led  to  their 
development  was  a  very  simple  one,  and  one  that  is 
frequently  before  us.  Koch  noticed  that  on  solid  sub- 
stances, such,  for  example,  as  a  slice  of  potato  or  of  moist 
bread,  which  had  been  exposed  for  a  time  to  the  air  and 
which  afforded  proper  nourishment  for  the  lower  organ- 
isms, there  developed  after  a  short  time  small  patches 
which  proved  to  be  colonies  of  bacteria.  Each  of 
these  colonies  on  closer  examination  proved  to  be,  as 
a  rule,  composed  of  distinct  species  of  micro-organisms. 
There  was  little  tendency  on  the  part  of  these  colonies 
to  become  confluent,  and  from  the  differences  in  their 
naked-eye  appearances  it  was  easy  to  see  that  they  rep- 
resented, in  the  main,  the  development  of  different 
species  of  bacteria. 

The  question  that  then  presented  itself  was  :  If  from 
a  mixture  of  organisms  floating  in  the  air  it  is  possible 
in  this  way  to  obtain  in  pure  cultures  the  component  in- 
dividuals, what  means  can  be  employed  for  obtaining  the 
7  97 


98  BA  CTERIOLOG  Y. 

same  results  at  will  from  mixture  of  different  species  of 
bacteria  when  found  together  under  other  conditions  ? 
It  was  plain  that  the  organisms  were  to  be  distinguished 
primarily,  the  one  from  the  other,  only  by  the  structure 
and  general  appearance  of  the  colonies  growing  from 
them,  for  by  their  morphology  alone  this  is  impossible. 
What  means  might  be  devised,  then,  for  separating  the 
individual  members  of  a  mixture  in  such  a  way  that 
they  would  remain  in  a  fixed  position,  and  be  so  widely 
separated,  the  one  from  the  other,  as  not  to  interfere 
with  the  production  of  colonies  of  characteristic  appear- 
ance, which  would,  under  favorable  conditions,  develop 
from  each  individual  cell? 

If  one  take  in  the  hand  a  mixture  of  barley,  rye, 
corn,  oats,  etc.,  and  attempt  to  separate  the  mass  into 
its  constituents  by  picking  out  the  different  grains,  the 
task  is  tedious,  to  say  the  least  of  it ;  but  if  the  handful 
of  grain  be  thrown  upon  a  large  flat  surface,  as  upon  a 
table,  the  grains  become  widely  separated  and  the  matter 
is  considerably  simplified ;  or,  if  sown  upon  proper  soil, 
the  various  grains  will  develop  into  growths  of  entirely 
different  external  appearance,  by  which  they  can  readily 
be  recognized  as  unlike  in  nature.  Similarly,  if  a  test- 
tube  of  decomposed  bouillon  be  poured  upon  a  large, 
flat  surface,  the  individual  bacteria  in  the  mass  are 
much  more  widely  separated,  the  one  from  the  other, 
than  they  were  when  the  bouillon  was  in  the  tube  ;  but 
they  are  in  a  fluid  medium,  and  there  is  no  possibility 
of  their  either  remaining  separated  or  of  their  forming 
colonies  under  these  conditions,  so  that  it  is  impossible 
by  this  means  to  pick  out  the  individuals  from  the 
mixture. 

If,  however,  some  substance  could  be  found  which 


METHODS  OF  ISOLATION. 


99 


possesses  the  property  of  being  at  one  time  fluid  and 
at  another  time  solid,  and  which  could  be  added  to 
this  bouillon  without  in  any  way  interfering  with  the 
life-functions  of  the  bacteria,  then,  as  solidification  set 
in,  the  organisms  would  be  fixed  in  their  positions,  and 
the  conditions  would  be  analogous  to  those  seen  on  the 
bit  of  potato. 

FIG.  14. 


Showing  certain  macroscopic  characteristics  of  colonies.    Natural  size. 

Gelatin  possesses  this  property,  and  it  was,  therefore, 
used.  At  a  temperature  which  does  not  interfere  with 
the  life  of  the  organisms  it  is  quite  fluid,  whereas  when 
subjected  to  a  lower  temperature  it  solidifies.  When 


1 00  BACTERIOLOG  Y. 

once  solid  it  may  be  kept  at  a  temperature  favorable  to 
the  growth  of  the  baeteria  and  will  remain  in  its  solid 
state. 

Gelatin  was  added  to  the  fluids  containing  mixtures 
of  bacteria,  and  the  whole  was  then  poured  upon  a  large, 
flat  surface,  allowed  to  solidify,  and  the  results  noted. 
It  was  found  that  the  conditions  seen  on  the  slice  of 
potato  could  be  reproduced  ;  that  the  individuals  in  the 
mixture  of  bacteria  grew  well  in  the  gelatin,  and,  as  on 
the  potato,  grew  in  colonies  of  typical  macroscopic  pecu- 
liarities, so  that  they  could  easily  be  distinguished  the 
one  from  the  other  by  their  naked-eye-appearances.  (See 
Fig.  14.)  It  was  necessary,  however,  to  use  a  more 
dilute  mixture  of  bacteria  than  the  original  decom- 
posed bouillon.  The  number  of  individuals  in  the 
tube  was  so  enormous  that  on  the  gelatin  plate  they 
were  so  closely  packed  together  that  it  was  impossible 
to  pick  them  out,  not  only  because  of  their  proximity 
the  one  to  the  other,  but  also  because  this  packing 
together  materially  interfered  with  the  production  of 
those  characteristic  differences  visible  to  the  naked 
eye.  The  numbers  of  the  organisms  were  then  dimin- 
ished by  a  process  of  dilution,  consisting  of  trans- 
ferring a  small  portion  of  the  original  mixture  into  a 
second  tube  of  sterilized  bouillon  to  which  gelatin  had 
been  added  and  liquefied ;  from  this  a  portion  was 
added  to  a  third  gelatin-bouillon  tube,  and  so  on. 
These  were  then  poured  upon  large  surfaces  and  allowed 
to  solidify.  The  result  was  entirely  satisfactory.  On 
the  gelatin  plates  from  the  original  tube,  as  was  ex- 
pected, the  colonies  were  too  numerous  to  be  of  use ; 
on  the  plates  made  from  the  first  dilution  they  were 
much  fewer  in  number,  but  usually  they  were  still  too 


METHODS  OF  I 


numerous  and  too  closely  packed  to  permit  of  charac- 
teristic growth  ;  on  the  second  dilution  they  were,  as  a 
rule,  fewer  in  number  and  widely  separated,  so  that 
the  individuals  of  each  species  were  in  no  way  pre- 
vented by  the  proximity  of  their  neighbors  from  grow- 
ing each  in  its  typical  way.  (See  Fig.  15.)  There 
was  then  no  difficulty  in  picking  out  the  colonies  result- 
ing from  the  growth  of  the  different  individual  bacteria. 


FIG.  15. 


ABC 

Series  of  plates  showing  the  results  of  dilution  upon  the  number  of  colonies : 
A.  Plate  No.  1,  or  "  original."  B.  First  dilution,  or  Plate  No.  2.  C.  Second 
dilution,  or  Plate  No.  3.  About  one-fourth  natural  size. 

This,  then,  is  the  principle  underlying  Koch's  method 
for  the  isolation  of  bacteria  by  means  of  solid  media. 

The  fundamental  constituent  of  the  media  employed 
is  the  bouillon,  which  contains  all  the  elements  necessary 
for  the  nutrition  of  most  bacteria,  the  gelatin  being  em- 
ployed simply  for  the  purpose  of  rendering  the  bouillon 
solid.  Tfee  medium  on  which  the  organisms  are  grow- 
ing is,  therefore,  simply  solidified  bouillon,  or  beef- 
tea. 

In  practice,  gelatinous  substances  are  employed — the 
one  an  animal  or  bone  gelatin,  the  ordinary  table  gelatin 
of  good  quality ;  the  other  a  vegetable  gum,  known 
us  agar-agar,  the  native  name  for  Ceylon  moss  or  Ben- 


'  BACTERIOLOGY. 

gal  isinglass,  which  is  obtained  from  a  group  of  algae 
growing  in  the  sea  along  the  coast  of  Japan,  China,  and 
many  parts  of  the  East,  where  it  is  employed  as  an 
article  of  diet  by  the  natives. 

The  behavior  of  the  two  gelatinous  substances  under 
the  influence  of  heat  and  of  bacterial  growth  renders  them 
of  different  application  in  bacteriological  work.  The 
animal  gelatin  liquefies  at  a  much  lower  temperature, 
and  also  requires  a  lower  temperature  for  its  solidifica- 
tion, than  does  the  agar-agar.  Ordinary  gelatin,  in  the 
proportion  commonly  used  in  this  work,  liquefies  at 
about  24°-26°  C.,  and  becomes  solid  at  from  8°  to  10°  C. 
It  may  be  employed  for  those  organisms  which  do  not 
require  a  higher  temperature  for  their  development  than 
22°-24°  C.  Agar-agar,  on  the  other  hand,  does  not 
liquefy  until  the  temperature  has  reached  about  98°-99° 
C.  It  remains  fluid  ordinarily  until  the  temperature  has 
fallen  to  38°-39°  C.,  when  it  rapidly  solidifies.  For 
our  purposes,  only  that  form  of  agar-agar  can  be  used 
which  remains  fluid  at  from  38°  to  40°  C.  Agar-agar 
which  remains  fluid  only  at  a  temperature  above  this 
point  would  be  too  hot,  when  in  a  fluid  state,  for  use ; 
many  of  the  organisms  introduced  into  it  would  either 
be  destroyed  or  checked  in  their  development  by  so  high 
a  temperature.  Agar-agar  is  employed  in  those  cases  in 
which  the  cultivation  must  be  conducted  at  a  tempera- 
ture above  the  melting-point  of  gelatin. 

In  addition  to  their  thermal  reactions,  these  two 
gelatinous  substances  are  aifected  very  differently  by 
different  species  of  bacteria.  As  we  shall  learn  later, 
certain  bacteria  elaborate  in  the  course  of  their  growth 
digestive  (proteolytic)  enzymes  or  ferments  that,  in  their 
action  upon  proteid  matters,  are  strikingly  like  pepsin 


METHODS  IN  ISOLATION.  103 

in  some  and  trypsin  in  other  instances.  When  bacteria 
endowed  with  this  physiological  property  are  cultivated 
upon  bone  gelatin  their  growth  is  accompanied  by  the 
progressive  digestion  (liquefaction)  of  the  gelatin,  which 
liquefied  gelatin  cannot  again  be  brought  to  a  solid  con- 
dition. We  know  of  no  bacteria  capable  of  producing 
a  similar  liquefaction  of  agar-agar  or  vegetable  gum. 
This  striking  difference  between  the  two  gelatinous  sub- 
stances under  the  influence  of  bacterial  activity  is  one 
of  the  most  important  and  commonly  employed  differ- 
ential reactions  in  the  identification  of  species. 

As  a  rule,  the  colony-formations  seen  upon  gelatin 
are  much  more  characteristic  than  those  which  develop 
on  agar-agar,  and  for  this  reason  gelatin  is  to  be  pre- 
ferred when  circumstances  will  permit.  Both  gelatin 
and  agar-agar  may  be  used  in  the  preparation  of  plates 
and  Esmarch  tubes,  subsequently  to  be  described. 


CHAPTER  V. 

Preparation    of   media — Bouillou,    gelatin,    agar-agar,   potato,   l>loo<l- 
serum,  etc. 

As  has  been  stated,  the  fundamental  constituent  of 
culture-media  is  beef-tea,  or  bouillon. 

BOUILLON. — The  directions  of  Koch  for  the  prepara- 
tion of  this  medium  have  undergone  many  modifications 
to  meet  special  cases  ;  but  for  general  use  his  original 
formula  is  still  retained.  It  is  as  follows  :  500  grammes 
of  finely  chopped  lean  beef,  free  from  fat  and  tendons, 
are  to  be  soaked  jn  one  litre  of  water  for  twenty-four 
hours,  during  which  time  the  mixture  is  to  remain  in 
an  ice-chest  or  to  be  otherwise  kept  at  a  low  tempera- 
ture. At  the  end  of  twenty-fogr  hours  it  is  to  be  strained 
through  a  coarse  towel  and  pressed  until  a  litre  of  fluid  is 
obtained.  To  this  are  to  be  added  10  grammes  (1,0  per 
cent.)  of  dried  peptone  and  5  grammes  (0.5  per  cent.)  of 
common  salt  (NaCl).  It  is  then  to  be  rendered  exactly 
neutral  or  very  slightly  alkaline  to  litmus  with  a  few 
drops  of  saturated  sodium  carbonate  solution.  The  flask 
containing  the  mixture  is  then  to  be  placed  either  in  a 
steam  sterilizer  or  in  a  wrater-bath,  or  over  a  free  flame, 
and  kept  at  the  boiling-point  until  all  the  albumin  is 
coagulated  and  the  fluid  portion  is  of  a  clear,  pale 
straw  color.  It  is  then  filtered  through  a  folded  paper 
filter  and  sterilized  in  the  steam  sterilizer  by  the  frac- 
tional method.  Certain  modifications  of  this  method 
are  of  sufficient  value  to  justify  mention.  Most  im- 

104 


BOUILLON.  105 

portant  is  the  neutralization.  Ordinarily,  this  is  ac- 
complished with  the  saturated  sodium  carbonate  solu- 
tion, and  the  reaction  is  determined  with  red  and  blue 
litmus  papers  ;  for  the  beginner  this  method  serves  most 
purposes. 

The  sodium  carbonate  solution  is  not  so  good,  how- 
ever, as  a  strong  solution  of  caustic  soda  or  potash, 
because  the  carbonic  acid  liberated  from  the  sodium 
carbonate  frequently  gives  rise  to  a  confusing,  tem- 
porary acid  reaction  which  disappears  on  heating; 
nor  is  litmus  the  most  reliable  indicator  to  employ. 
To  obviate  this,  Schultz1  recommends  exact  titration 
with  a  solution  of  caustic  soda.  For  this  purpose  a  4 
per  cent,  solution  of  caustic  soda  is  prepared.  From  this 
a  0.4  per  cent,  solution  is  made,  and  with  it  the  titration 
is  practised.  After  the  bouillon  has  been  deprived  of  all 
coagulable  albumin  and  blood-coloring-matter  by  boiling 
and  nitration,  and  has  cooled  down  to  the  temperature 
of  the  air,  its  volume  is  exactly  measured. 

From  this  a  sample  of  exactly  5  or  10  c.c.  is  then  taken, 
and  to  it  a  few  drops  of  one  of  the  indicators  com- 
monly employed  in  analytical  work  are  added.  Schultz 
recommends  1  drop  of  phenolphtalein  solution  (1 
gramme  of  phenolphtalein  in  300  c.c.  of  alcohol)  to  1 
c.c.  of  bouillon.  The  beaker  containing  the  sample  is 
placed  upon  white  paper,  and  the  dilute  caustic  soda 
solution  is  then  allowed  to  drop  very  slowly  into  it 
from  a  burette,  until  there  appears  a  very  delicate  rose 
color,  which  indicates  the  beginning  of  alkaline  reaction. 
A  second  sample  of  the  bouillon  is  treated  in  the  same 
way.  If  the  amounts  of  caustic  soda  solution  required 

1  Schultz:  Centralbl.  f.  Bakt.  u.  Parasitenkunde,  1891,  vol.   x.  Nos. 
2  and  3. 


106  BA  CTERIOL  OGY. 

for  each  sample  deviate  but  very  slightly  or  not  at  all 
the  one  from  the  other,  the  mean  of  these  amounts  is 
taken  as  the  amount  of  alkali  necessary  to  neutralize 
the  quantity  of  bouillon  employed.  If  10  c.c.  of  bouillon 
were  employed,  then  for  the  whole  amount  of  1  litre 
just  100  times  as  much,  minus  that  for  the  two  samples 
used  in  titration,  will  be  needed.  For  example :  to 
neutralize  10  c.c,  of  bouillon  2  c.c.  of  the  diluted  (0.4 
per  cent.)  caustic  soda  solution  were  employed.  For 
the  remaining  980  c.c.  of  the  litre  of  bouillon,  then, 
196  c.c.  (200  c.c.  less  4  c.c.,  the  amount  employed  for  the 
two  samples  of  10  c.c.  each  of  bouillon)  are  needed  of 
the  0.4  per  cent,  solution,  or  one-tenth  of  this  amount 
of  the  4  per  cent,  caustic  soda  solution. 

For  the  neutralization  of  the  whole  bulk*T>f  the 
bouillon  it  is  better  to  employ  the  stronger  alkaline 
solution,  as  by  its  use  the  volume  is  not  increased 
to  so  great  an  extent  as  when  the  dilute  solution  is 
used. 

It  is  evident  that  this  method  is  much  more  exact 
than  that  ordinarily  employed  ;  but  at  the  same  time  it 
must  be  remembered  that  for  its  success  exactness  in 
the  measurement  of  the  volumes  and  in  the  preparation 
of  the  dilutions  is  required.  To  obviate  error,  it  is 
better  to  employ  this  method  when  the  solutions  are  all 
cool  and  of  nearly  the  same  temperature,  so  that  rapid 
fluctuations  in  temperature,  and  consequent  alterations 
in  volume,  will  not  materially  interfere  with  the  accu- 
racy of  the  results. 

This  method  of  neutralization,  as  suggested  by 
Schultz.  should  be  adopted  for  experiments  in  which  it 
is  desirable  to  have  the  reaction  of  the  medium  accurate 
and  constantly  of  the  same  degree. 


BOUILLON.  107 

For  the  purposes  of  the  beginner,  however,  results 
quite  satisfaetory  in  their  nature  may  be  obtained  by  the 
employment  of  the  saturated  sodium  carbonate  solution 
for  neutralization,  with  litmus  paper  as  the  indicator. 

In  the  exhaustive  paper  of  Fuller l  on  the  question  of 
reaction  it  was  shown  that  the  results  obtained  by  titrating 
the  same  culture-medium  with  the  same  alkaline  solu- 
tion diifered  very  markedly  with  the  indicator  employed. 
For  instance,  a  litre  of  ordinary  meat-infusion  nutrient 
agar-agar  required  47  c.c.  of  a  normal  caustic  alkali 
solution  to  neutralize  it  when  phenolphtalein  was  the 
indicator  used,  28  c.c.  when  blue  litmus  was  employed, 
and  5  c.c.  when  rosolic  acid  was  substituted.  It  is 
manifest  from  this  that  the  actual  reactions  of  media, 
in  the  neutralization  of  which  different  indicators  have 
been  used,  may  differ  very  widely  from  one  another, 
and  that  the  results  of  cultivation  on  a  medium  neu- 
tralized by  one  method  are  not  fairly  comparable  with 
those  obtained  when  another  indicator  has  been  used. 
For  the  sake  of  uniformity  Fuller  suggests  that  bac- 
teriologists should  agree  upon  some  one  trustworthy 
method  of  neutralization  and  employ  it  to  the  exclusion 
of  other  methods.  He  recommends,  as  the  procedure 
that  has  given  the  most  satisfactory  results  in  his  hands, 
a  modification  of  Schultz's  method,  viz.,  5  c.c.  of  the 
culture-medium  are  to  be  mixed  with  45  c.c.  of  distilled 
water  in  a  porcelain  evaporating-dish  and  boiled  for 
three  minutes,  after  which  1  c.c.  of  phenolphtalein 
solution 2  is  added  and  titration  with  the  one-twentieth 
normal  caustic  alkali  solution  is  quickly  made.  The 

1  Fuller:  "On  the  Proper  Eeaction  of  Nutrient  Media  for  Bacterial 
Cultivation,"  Public  Health  (Journal  of  the  American  Public  Health 
Association),  Quarterly  Series,  1895,  vol.  i.  p.  381. 

*  A  0.5  per  cent,  solution  of  the  powder  in  50  per  cent,  alcohol. 


108  J3A  CTERIOLOG  Y. 

neutral  point  (slightly  on  the  side  of  alkalinity)  is  indi- 
cated by  the  appearance  of  a  pink  color,  the  effect  of 
the  alkali  on  the  phenolphtalein.  From  the  amount 
of  one-twentieth  normal  alkali  solution  needed  for  5 
c.c.  of  the  medium  it  is  easy  to  calculate  the  number »of 
cubic  centimetres  of  the  normal  solution  that  will  be 
required  to  neutralize  the  entire  mass. 

The  phenolphtalein  neutral  point  lies  so  high,  aver- 
aging 47  c.c  of  normal  caustic  alkali  solution  per  litre 
for  nutrient  meat-infusion  agar-agar,  and  56  c.c.  pel- 
litre  for  nutrient  gelatin,  that  it  is  improbable  from  ex- 
perience gained  from  the  older  methods  that  the  condi- 
tions offered  by  media  neutral  to  this  indicator  are  suit- 
able for  the  growth  of  all  bacteria,  so  that  with  particular 
species  it  may  be  necessary  to  determine  by  experiment 
the  degree  of  deviation  from  the  neutral  point  that  is 
best  suited  for  development.  In  Fuller's  experience  the 
degree  of  deviation  from  the  phenolphtalein  neutral 
point  that  gives  in  general  the  best  results  is  represented 
by  from  15  to  20  of  his  scale — i.  e.,  there  should  remain 
enough  uncombined  acid  in  a  litre  of  the  finished  me- 
dium to  require  the  further  addition  of  caustic  alkali  to 
the  extent  of  from  15  to  20  c.c.  of  a  normal  solution  to 
bring  the  reaction  of  the  mass  up  to  the  phenolphtalein 
neutral  point.  Thus,  for  example,  if  upon  titration  it 
should  be  found  that  to  neutralize  a  litre  of  nutrient 
meat-infusion  gelatin  by  the  phenolphtalein  process 
55  c.c.  of  normal  caustic  alkali  solution  would  be 
needed,  the  amount  actually  added  would  be  from  35 
to  40  c.c. — i.  e.9  from  15  to  20  c.c.  less  than  the 
amount  needed  to  bring  the  reaction  up  to  the  neutral 
point. 

Not   infrequently   the   filtered   bouillon,    neutralized 


NUTRIENT  GELATIN.  109 

and  sterilized,  will  be  seen  to  contain  a  fine,  flocculent 
precipitate.  This  may  be  due  either  to  excess  of  alka- 
linity or  to  incomplete  precipitation  of  the  albumin. 
The  former  may  be  corrected  with  dilute  acetic  or 
hydrochloric  acid,  and  the  bouillon  again  boiled,  fil- 
tered, and  sterilized  ;  or,  if  due  to  the  latter  cause,  sub- 
sequent boiling  and  nitration  usually  result  in  ridding 
the  bouillon  of  the  precipitate. 

Another  modification  now  generally  employed  is 
the  use  of  meat-extracts  instead  of  infusion  of  meat. 
Almost  any  of  the  meat-extracts  of  commerce  answer 
the  purpose,  though  we  usually  employ  Liebig's.  It 
is  used  in  the  strength  of  from  two  to  four  grammes  to 
the  litre  of  water.  Peptone  and  sodium  chloride  are 
added  as  in  the  bouillon  made  from  meat-infusion. 
The  advantages  of  meat-extract  are  :  it  takes  less  time ; 
affords  a  solution  of  more  uniform  composition  if  used 
in  fixed  proportions ;  and  in  general  use  gives  results 
that  are  equally  as  satisfactory  as  those  obtained  from 
the  employment  of  infusion  of  meat. 

NUTKIENT  GELATIN. — For  the  preparation  of  gelatin 
the  bouillon  is  first  made  in  the  way  given,  except 
that  its  reaction  is  corrected  after  the  gelatin  has 
been  completely  dissolved,  which  occurs  very  rapidly 
in  hot  bouillon.  The  reaction  of  the  gelatin  of  com- 
merce is  frequently  quite  acid,  so  that  a  much  larger 
amount  of  alkali  is  needed  for  its  neutralization  than 
for  other  media.  It  is  possible,  however,  to  obtain 
from  the  makers  an  excellent  grade  of  gelatin  from 
which  all  acid  has  been  carefully  washed.1  The  gelatin 
is  added  in  the  proportion  of  10  to  12  per  cent.  Its 

1  Hesteberg's  acid-free,  gold-label  gelatin  has  given  us  entire  satis- 
faction in  this  respect.     It  is  an  imported  article. 


110  BA  CTERIQL  0  G  Y. 

complete  solution  may  be  accomplished  either  over  a 
water-bath,  in  the  steam  sterilizer,  or  over  a  free  flame. 
If  the  latter  method  be  practised,  care  must  be  taken 
that  the  mixture  is  constantly  stirred  to  prevent  burn- 
ing at  the  bottom  and  consequent  breaking  of  the  flask, 
if  a  flask  is  employed. 

It  is  now  almost  the  universal  practice  to  use  enam- 
elleled  iron  saucepans,  instead  of  glass  flasks,  for  the 
purpose  of  making  both  gelatin  and  agar-agar ;  by  this 
means  the  free  flame  may  be  employed  without  danger 
of  breaking  the  vessel,  and,  with  a  little  care,  without 
burning  the  media.  Under  any  conditions  it  is  better 
to  protect  the  bottom  of  the  vessel  from  the  direct 
action  of  the  flame  by  the  interposition  of  several  layers 
of  wire  gauze,  a  thin  sheet  of  asbestos-board,  or  an  ordi- 
nary cast-iron  stove-plate. 

When  the  gelatin  is  completely  melted  it  may  be 
filtered  through  a  folded  paper  filter  supported  on  an 
ordinary  funnel ;  if  solution  is  complete,  this  should  be 
very  quickly  accomplished. 

For  the  filtration  of  such  substances  as  gelatin  and 
agar-agar  it  is  of  much  importance  to  have  a  properly 
folded  filter.  Inability  to  fold  a  filter  properly  is  so 
common  with  beginners  that  a  detailed  description  of 
the  steps  may  not  be  out  of  place.  To  fold  a  filter  cor- 
rectly, proceed  as  follows  :  a  circular  piece  of  filter 
paper  is  folded  exactly  through  its  centre,  forming  the 
fold  1,1'  (Fig.  16) ;  the  end  1  is  then  folded  over  to  V, 
forming  the  fold  5;  1  and  V  are  each  then  brought  to 
5,  thus  forming  the  folds  3  and  7 ;  1  is  then  carried  to 
the  point  7,  and  the  fold  4  is  formed,  and  by  carrying 
V  to  3  the  fold  6  is  produced  ;  and  by  bringing  1  to  3 
and  V  to  7  the  folds  2  and  8  result, 


NUTRIENT  GELATIN. 


Ill 


Thus  far  the  ridges  of  all  folds  are  on  the  side  of  the 
paper  next  to  the  table  on  which  we  are  folding.     The 


paper  is  now  taken  up  and  each  space  between  the  seams 
just  produced  is  to  be  subdivided  by  a  seam  or  fold 
through  its  centre,  as  indicated  by  the  dotted  lines  in 
Fig.  16,  but  with  the  creases  on  the  side  opposite  to  that 


FIG.  17. 


occupied  by  the  creases  1,  2,  3,  4,  etc.,  first  made.  As 
each  of  these  folds  is  made  the  paper  is  gradually  folded 
into  a  wedge-shaped  bundle  (Fig.  17,  a),  which  when 
opened  assumes  the  form  of  a  properly  folded  filter 
(seen  in  6,  Fig.  17).  Before  placing  it  upon  the  fun- 
nel it  is  well  to  go  over  each  crease  and  see  that  it  is 
as  closely  folded  as  possible,  care  being  taken  not  to 
tear  it.  The  advantage  of  the  folded  filter  is  that  by 


112  BA  CTERIOL  OG  Y. 

its  use  a  much  greater  filtering  surface  is  obtained,  as  it 
is  in  contact  with  the  funnel  only  at  the  points  formed 
by  the  ridges,  leaving  the  greater  part  of  the  flat  surface 
free  for  nitration. 

The  employment  of  the  hot- water  funnel,  so  often 
recommended,  has  been  dispensed  with  in  this  work  to 
a  very  large  extent,  for  the  reason  that  if  solution  of 
the  gelatin  is  complete,  nitration  is  so  rapid  as  not  to 
necessitate  the  use  of  an  apparatus  for  maintaining  a 
high  temperature.  The  temperature  at  which  the  hot- 
water  funnel  retains  the  gelatin  is  so  high  that  evapora- 
tion and  concentration  rapidly  occur,  and  in  consequence 
nitration  is,  as  a  rule,  retarded.  The  nitration  is  fre- 
quently done  in  the  steam  sterilizer ;  but  this  too  is 
unnecessary  if  the  gelatin  is  quite  dissolved.  At  the 
ordinary  temperature  of  the  room,  and  by  the  means 
commonly  employed  for  the  nitration  of  other  sub- 
stances, both  gelatin  and  agar-agar  may  be  rapidly 
filtered  if  they  are  completely  dissolved. 

It  not  infrequently  occurs  that,  even  under  the  most 
careful  treatment,  the  filtered  gelatin  is  not  perfectly 
transparent  (the  condition  to  which  it  must  be  brought, 
otherwise  it  is  useless),  and  clarification  becomes  neces- 
sary. For  this  purpose  the  mass  must  be  redissolved, 
and  when  at  a  temperature  between  60°  and  70°  C.  an 
egg,  which  has  been  beaten  up  with  about  50  c.c.  of 
water,  is  added.  The  whole  is  then  thoroughly  mixed 
together  and  again  brought  to  the  boiling-point,  and 
kept  there  until  coagulation  of  the  albumin  occurs.  The 
albumin  coagulates  as  large  flocculent  masses,  and  it  is 
better  not  to  break  them  up  if  it  can  be  avoided,  as 
when  broken  up  into  fine  flakes  they  clog  the  filter  and 
materially  retard  filtration. 


NUTRIENT  GELATIN.  113 

The  practice  sometimes  recommended  of  removing 
these  albuminous  coagula  by  first  filtering  the  gelatin 
through  a  cloth,  and  then  through  paper,  is  not  only 
superfluous,  but  in  most  instances  renders  the  process 
of  filtration  much  more  difficult,  because  of  the  disin- 
tegration of  the  masses  into  finer  particles,  which  have 
the  effect  just  mentioned,  viz.,  of  clogging  the  filter. 

Under  no  circumstances  should  a  filter  be  used  with- 
out first  having  been  moistened  with  water.  If  this  is  not 
done,  the  pores  of  the  paper,  which  are  relatively  large 
when  in  a  dry  state,  when  moistened  by  the  gelatin  not 
only  diminish  in  size,  but  in  contracting  are  often  en- 
tirely occluded  by  the  finer  albuminous  flakes  which 
become  fixed  within  them,  and  filtration  practically 
ceases.  The  preliminary  moistening  with  water  causes 
diminution  of  the  size  of  the  pores  to  such  an  extent 
that  the  finer  particles  of  the  precipitate  rest  on  the 
surface  of  the  paper,  instead  of  becoming  fixed  in  its 
meshes. 

During  boiling  it  is  well  to  filter,  from  time  to  time, 
a  few  cubic  centimetres  of  the  gelatin  into  a  test-tube 
and  boil  it  over  a  free  flame  for  a  minute  or  so  ;  in  this 
way  one  can  detect  if  all  the  albumin  has  been  coagu- 
lated— i.  e.,  if  the  solution  is  ready  for  filtration. 

Gelatin  should  not,  as  a  rule,  be  boiled  more  than 
ten  or  fifteen  minutes  at  one  time,  or  be  left  in  the 
steam  sterilizer  for  more  than  thirty  minutes;  other- 
wise its  property  of  solidifying  may  be  impaired. 

As  soon  as  the  preparation  of  the  gelatin  is  complete, 
whether  it  is  retained  in  the  flask  into  which  it  has 
IKVII  filtered  or  decanted  into  sterilized  test-tubes,  it 
should  be  sterilized,  the  mouth  of  the  flask  or  the  test- 
tubes  containing  it  having  previously  been  closed  with 


114  BACTERIOLOGY. 

cotton  plugs.  It  may  be  sterilized  by  either  the  inter- 
mittent method  with  streaming  steam  or  by  a  single 
application  of  steam  under  pressure  in  the  autoclave. 
If  the  latter  method  be  selected,  the  pressure  should 
not  exceed  one  atmosphere  and  the  time  of  exposure  be 
not  over  fifteen  minutes. 

NUTRIENT  AGAR-AGAR. — The  preparation  of  nutrient 
agar-agar  by  the  beginner  is  far  too  frequently  a  tedious 
and  time-consuming  operation.  This  is  due  mainly  to 
lack  of  patience  and  to  deviation  from  the  rules  laid 
down  for  the  preparation  of  this  medium.  If  the 
directions  given  below  for  the  preparation  of  nutrient 
agar-agar  be  strictly  observed,  no  difficulty  whatever 
should  be  encountered.  Many  methods  are  recom- 
mended for  its  preparation,  almost  every  worker  having 
some  slight  modification  of  his  own. 

The  methods  that  have  given  us  the  best  results,  and 
from  which  we  have  no  good  grounds  for  departing, 
are  as  follows : 

Prepare  the  bouillon  in  the  usual  way.  Agar-agar 
reacts  neutral  or  very  slightly  alkaline,  so  that  the 
bouillon  may  be  neutralized  before  the  agar-agar  is 
added.  Then  add  finely  chopped  or  powdered  agar- 
agar  in  the  proportion  of  1  to  1.5  per  cent.  Place  the 
mixture  in  a  porcelain-lined  iron  vessel,  and  on  the  side 
of  the  vessel  make  a  mark  at  the  height  at  which  the  level 
of  the  fluid  stands ;  if  a  litre  of  medium  is  being  made, 
add  about  250  to  300  c.c.  more  of  water  and  allow  the 
mass  to  boil  slowly,  occasionally  stirring,  over  a  free 
flame,  from  one  and  a  half  to  two  hours ;  or  until 
the  excess  of  water — i.  e.,  the  250  or  300  c.c.  that 
were  added — has  evaporated.  Care  must  be  taken 
that  the  mixture  does  not  boil  over  the  sides  of  the 


NUTRIENT  AGAR-AGAR.  115 

vessel.  From  time  to  time  observe  if  the  fluid  has 
fallen  below  its  original  level ;  if  it  has,  add  water 
until  its  Volume  of  1  litre  is  restored.  At  the  end  of 
the  time  given  remove  the  flame  and  place  the  vessel 
containing  the  mixture  in  a  large  dish  of  cold  water ; 
stir  the  agar-agar  continuously  until  it  has  cooled  to 
about  68°-70°  C.,  and  then  add  the  white  of  one  egg 
which  has  been  beaten  up  in  about  50  c.c.  of  water ;  or 
the  ordinary  dried  albumin  of  commerce  may  be  dis- 
solved in  cold  water  in  the  proportion  of  about  10  per 
cent,  and  used  ;  the  results  are  equally  as  good  as  wThen 
eggs  are  employed.  Mix  this  carefully  throughout  the 
agar-agar  and  allow  the  mass  to  boil  slowly  for  about 
another  half-hour,  observing  all  the  while  the  level  of 
the  fluid,  which  should  not  fall  below  the  litre  mark. 
It  is  necessary  to  reduce  the  temperature  of  the  mass 
to  the  point  given,  68°-70°  C.,  otherwise  the  coagula- 
tion of  the  albumin  will  occur  suddenly  in  lumps  and 
masses  as  soon  as  it  is  added,  and  its  clarifying  action 
will  not  be  uniform.  The  process  of  clarification 
with  the  egg  is  purely  mechanical ;  the  finer  particles, 
which  would  otherwise  pass  through  the  pores  of  the 
filter,  being  taken  up  by  the  albumin  as  it  coagulates 
and  being  retained  in  the  coagula. 

At  the  end  of  one-half  hour  the  boiling  mass  may 
be  easily  and  quickly  filtered  through  a  heavy,  folded 
paper  filter  at,  the  room-temperature;  as  a  rule,  the 
filtrate  is  as  clear  and  transparent  as  agar-agar  usually 
appears. 

It  may  be  well  to  emphasize  the  fact  that  for  the 
filtration  of  agar-agar  a  hot- water  funnel,  or  any  other 
special  device  for  maintaining  the  temperature  of  the 
mass,  is  entirely  unnecessary.  Agar-agar  prepared  after 


116  BACTERIOLOGY. 

the  methods  just  given  should  pass  through  a  properly 
folded  paper  filter  at  the  rate  of  a  litre  in  from  twelve 
to  fifteen  minutes. 

Another  plan  that  insures  complete  solution  of  the 
agar-agar  without  causing  the  precipitates  often  seen 
when  all  the  ingredients  are  added  at  once  and 
boiled  for  a  long  time,  is  to  weigh  out  the  necessary 
amount  of  agar-agar,  10  or  15  grammes,  and  place  this 
in  1300  or  1400  c.c.  of  water  and  boil  down  over  a  free 
flame  to  1000  c.c.  The  peptone,  salt,  and  beef-extract 
are  then  added  and  the  boiling  continued  until  they 
are  dissolved.  The  clarification  with  egg-albumin 
may  then  be  done,  and  usually  the  mass  filters  quite 
clear  and  does  not  show  the  presence  of  precipitates 
upon  cooling.  If  the  mixture  is  positively  alkaline,  it 
is  not  only  cloudy,  but  it  filters  with  difficulty  ;  if  it  is 
acid,  it  is  usually  quite  clear,  and  filters  more  quickly, 
but,  as  Schultz  has  pointed  out,  it  loses  at  the  same  time 
some  of  its  gelatinizing  properties.  The  bouillon  should 
always  be  neutralized  before  the  agar-agar  is  added  to 
it ;  for  if  the  bouillon  be  acid  from  the  juices  of  the 
meat,  it  robs  the  agar-agar,  under  the  influence  of 
heat,  of  part  of  its  gelatinizing  power,  which  will  not 
be  regained  by  subsequent  neutralization. 

Another  method  by  which  agar-agar  can  be  easily 
and  quickly  melted  is  by  steam  under  pressure.  If 
the  flask  containing  the  mixture  of  bouillon  and  agar- 
agar  be  kept  in  the  digester  or  autoclave  for  ten  minutes 
with  the  steam  under  a  pressure  of  about  one  atmos- 
phere, as  shown  by  the  gauge,  the  agar-agar  will  be 
found  at  the  end  of  this  time  completely  melted,  and  fil- 
tration may  then  be  accomplished  with  but  little  difficulty. 

If  glycerin  is  to  be  added  to  the  agar-agar,  it  is  done 


PREPARATION  OF  POTATOES.  117 

after  filtration  and  before  sterilization.  The  nutritive 
properties  of  the  media  for  certain  organisms,  particu- 
larly the  tubercle  bacillus,  are  increased  by  the  addition 
of  glycerin  in  the  proportion  of  5  to  7  per  cent. 

If  after  filtration  a  fine  flocculent  precipitate  is  seen, 
look  to  the  reaction  of  the  medium.  If  it  is  quite 
alkaline,  neutralize,  boil,  and  filter  again.  If  the 
reaction  is  neutral  or  only  very  slightly  acid,  dissolve 
and  again  clarify  with  egg-albumin  by  the  method 
given. 

The  most  important  feature  of  all  the  media,  aside 
from  the  correct  proportion  of  the  ingredients,  is  their 
reaction.  They  must  be  neutral  or  very  slightly  alkaline 
to  litmus.  (See  remarks  on  Neutralization  of  Media.) 
Only  a  few  organisms  develop  well  on  media  of  an  acid 
reaction.  In  all  of  the  media  mentioned  above  the  meat- 
extracts  now  on  the  market  may  usually  be  substituted 
for  the  meat  itself  in  preparing  the  bouillon.  They 
may  be  employed  in  the  proportion  of  from  two  to  four 
grammes  to  the  litre  of  water. 

PREPARATION  OF  POTATOES. — Potatoes  are  prepared 
for  use  in  two  ways : 

1.  They  are  taken  as  they  come  to  market — old 
potatoes  being  usually  recommended — and  carefully 
scrubbed  under  a  water-tap  with  a  stiff  brush  until 
all  adherent  dirt  has  been  removed  ;  "  the  eyes "  and 
all  discolored  or  decayed  parts  are  carefully  removed 
with  a  pointed  knife.  They  are  then  placed  in  a  solu- 
tion of  corrosive  sublimate  of  the  strength  of  1  : 1000, 
where  they  are  allowed  to  remain  for  twenty  minutos ; 
at  the  end  of  this  time,  without  rinsing  off  the  sublimate, 
they  are  placed  in  a  covered  tin  bucket  with  a  perforated 
bottom  and  sterilized  in  the  steam  sterilizer  for  forty- 


1 1 8  BA  CTERIOLOG  Y. 

five  minutes.  On  the  second  and  third  days  the  steril- 
ization is  repeated  for  fifteen  to  twenty  minutes  each  day. 
They  must  not  be  removed  from  the  sterilizing  bucket 
until  sterilization  is  complete,  when  they  are  ready  for 
use.  When  prepared  in  this  way  they  are  usually  in- 
tended to  be  cut  in  half,  and  the  cultivation  of  the 
organisms  is  to  be  conducted  upon  the  flat  surfaces 
of  the  sections.  (Koch's  original  method.) 

This  method  requires  some  care  to  prevent  contam- 
ination during  manipulation.  The  hand  which  is  to 
take  up  the  potato  from  the  bucket,  which  until  now  has 
remained  covered,  is  first  disinfected  in  the  sublimate 
solution  for  ten  minutes ;  the  potato  is  then  taken  up 
between  the  thumb  and  index  finger  and  severed  in 
two  by  a  knife  which  has  just  been  sterilized  in  a  free 
flame  until  it  is  quite  hot.  The  knife  is  passed  not 
quite  through  the  potato,  but  nearly  so.  A  large  glass 
culture-dish  for  the  reception  of  the  halves  of  the 
potato,  having  been  disinfected  for  twenty  minutes  with 
1  : 1000  sublimate  solution  and  then  drained  of  all  ad- 
herent solution,  should  be  at  hand  ready  for  the  potato ; 
the  cover  is  removed,  and  by  twisting  the  knife  gently 
the  halves  of  the  potato  may  be  caused  to  fall  apart  in 
the  dish  and  usually  to  fall  upon  their  convex  surfaces, 
leaving  the  flat  sections  uppermost.  The  cover  of 
the  dish  is  replaced  and  the  potatoes  are  ready  for 
inoculation. 

2.  Preparation  of potatoes  for  test-tube  cultures.  Method 
of  Bolton.1  If  the  potatoes  are  to  be  employed  for  test- 
tube  cultures,  one  simply  scrubs  off  the  coarser  particles 
of  dirt  with  water  and  a  brush,  and  with  a  cork- borer 
punches  out  cylindrical  bits  of  potato  which  will  fit 

1  Medical  News,  vol.  i.  of  1887,  No.  12,  p.  318. 


PREPARATION  OF  POTATOES. 


119 


FIG.  18. 


loosely  into  the  test-tubes  to  be  used.  On  each  bit  of 
potato  is  then  to  be  cut  a  slanting  surface  running  from 
about  the  junction  of  the  first  and  second  thirds  of  the 
cylinder  to  the  diagonally  opposite  end.  These  cylin- 
ders of  potato  are  to  be  left  in  running  water  over 
night,  otherwise  they  will  be  very  much  discolored  by  the 
sterilization  to  which  they  are  to  be  subjected.  After 
being  thus  washed  they  are  placed  in  pre- 
viously prepared  test-tubes,  one  piece  in  each 
tube,  with  the  slanting  surface  up,  the  cot- 
ton plugs  of  the  tubes  replaced,  and  they 
are  then  to  be  sterilized  in  steam  for  fifteen 
to  twenty  minutes  on  each  of  three  successive 
days.  Or  the  entire  sterilization  may  be  ac- 
complished in  the  autoclave,  with  the  steam 
under  a  pressure  of  one  atmosphere,  by  a 
single  exposure  of  twenty  to  twenty-five 
minutes.  Potatoes  thus  prepared  have  the 
appearance  seen  in  Fig.  18,  except  that  there 
is  no  growth  upon  the  surface  as  is  shown 
in  the  cut. 

For  some  purposes  potatoes  may  be  ad- 
vantageously peeled,  sliced  into  discs  of 
about  1  cm.  in  thickness,  and  placed  in 
small  glass  dishes  provided  with  covers, 
similar  to  the  ordinary  Petri  dishes.  The  Potato  in  test- 

•  tube. 

dish  and   its  contents   are    then    sterilized 
by  steam  in  the  usual  way  (method  suggested  by  von 
Esmarch).     By  this   plan   a  relatively  large  area   for 
cultivation  is  obtained. 

Potatoes  may  also  be  boiled,  or  steamed,  and  mashed, 
and  the  mass  placed  in  covered  dishes,  test-tubes,  or 
flasks,  and  sterilized.  By  this  method  one  obtains  in 


120  BACTERIOLOGY. 

the  mass  a  mean  of  the  composition  of  the  several  pota- 
toes, or  bits  of  potatoes,  used  in  making  it,  an  advan- 
tage where  uniformity  is  desired. 

Care  must  be  given  to  the  sterilization  of  potatoes, 
because  they  always  have  adhering  to  them  the  organ- 
isms commonly  found  in  the  ground,  the  spores  of  which 
are  among  the  most  resistant  known.  The  so-called 
potato  bacillus  is  one  of  this  group ;  it  is  an  organism 
which  is  not  infrequently  more  or  less  of  an  obstacle 
to  the  work  of  the  beginner. 

BLOOD-SERUM. — By  the  original  method  of  preparing 
blood-serum  a  great  many  precautions  were  taken  that 
have  been  found  unnecessary  to  the  success  of  the  more 
modern  plans. 

It  is  possible  to  collect  serum  from  small  animals  and 
in  small  quantities  under  such  precautions  that  it  is  per- 
haps not  contaminated ;  but,  ordinarily,  for  laboratory 
purposes  a  larger  quantity  is  needed,  so  that  slaughter- 
houses are  the  source  from  which  it  is  usually  obtained, 
and  here  a  certain  amount  of  contamination  is  unavoid- 
able, though  its  extent  may  be  limited  by  proper  pre- 
cautions. 

The  precautions  to  be  taken  at  the  slaughter-house  in 
the  collection  of  blood  and  the  preparation  of  serum  for 
culture  purposes  are  about  as  follows : 

The  animal  from  which  the  blood  is  to  be  collected 
should  be  drawn  up  to  the  ceiling  by  the  hind  legs ;  the, 
head  should  be  held  well  back,  and  with  one  pass  of  a 
very  sharp  knife  the  throat  should  be  cut.  The  blood 
which  spurts  from  the  severed  vessels  should  be  col- 
lected in  large  glass  jars  which  have  been  previously 
cleaned  and  disinfected,  and  all  traces  of  the  disin- 
fectant removed  with  alcohol  and,  finally,  with  ether. 


BLOOD-SERUM.  121 

The  latter  evaporates  very  quickly  and  leaves  the  jar 
quite  dry.  The  jars  should  be  provided  with  covers, 
which  close  hermetically ;  these,  too,  should  be  care- 
fully disinfected.  The  best  form  of  vessels  for  the 
purpose  is  the  large  museum-jar  of  about  one  gallon 
capacity,  which  closes  by  a  cover  that  can  be  tightly 
screwed  down  upon  a  rubber  joint.  From  two  such 
jarfuls  of  blood  one  can  recover  quite  a  large  quan- 
tity of  clear  serum,  ordinarily  from  500  to  700  c.c.  The 
jars  having  been  filled  with  blood,  their  covers  are  placed 
loosely  upon  them  and  they  are  allowed  to  stand  for 
about  fifteen  minutes  until  clotting  has  begun.  At  the 
end  of  this  time  a  clean  glass  rod  is  passed  around  the 
edges  of  the  surface  of  the  clot  to  break  up  any  adhe- 
sions to  the  side  of  the  jar  that  may  have  formed,  and 
which  would  prevent  the  sinking  of  the  clot  to  the 
bottom.  The  covers  are  then  replaced  and  tightly 
clamped  in  position,  and  with  as  little  agitation  as  pos- 
sible the  jars  are  placed  in  an  ice-chest,  where  they 
remain  for  twenty-four  to  forty-eight  hours.  The 
temperature  should,  however,  not  be  low  enough  to 
prevent  coagulation,  but  should  be  sufficiently  low  to 
interfere  with  the  development  of  any  living  organ- 
isms that  may  be  present.  The  temperature  of  the 
ordinary  domestic  refrigerator  is  sufficient  for  the 
purpose.  After  twenty-four  to  forty-eight  hours  the 
clot  will  have  become  firm,  and  will  be  seen  at  the 
bottom  of  the  jar.  Above  it  is  a  quantity  of  dark 
straw-colored  serum.  The  serum  may  then  be  drawn 
off  with  a  sterilized  pipette  and  placed  in  tall  cylinders 
that  have  previously  been  plugged  with  cotton  wadding 
and  sterilized.  After  treating  all  the  serum  in  this  way, 
care  having  been  taken  to  exclude  as  much  as  possible  of 


122  BA  CTERIOL  OOY. 

the  coloring-matter  of  the  blood,  it  may  be  placed  again 
in  the  ice-chest  for  twenty-four  hours,  during  which 
time  the  corpuscular  elements  will  sink  to  the  bottom, 
leaving  the  supernatant  fluid  quite  clear.  This  may 
then  be  pipetted  off,  either  into  sterilized  test-tubes> 
about  8  c.c.  to  each  tube,  or  into  small  sterilized  flasks 
of  about  100  c.c.  capacity.  It  is  then  to  be  sterilized 
by  the  intermittent  method  at  low  temperatures,  viz.,  for 
one  hour  on  each  of  five  consecutive  days  at  a  tempera- 
ture of  68°-70°  C.  During  the  intervening  days  it  is 
to  be  kept  at  the  room-temperature  to  permit  of  the 
development  of  any  spores  that  may  be  present  into 
their  vegetative  forms,  in  which  condition  they  are 
killed  by  an  hour's  exposure  to  the  temperature  of 
70°  C. 

FIG.  19. 


Chamber  for  sterilizing  and  solidifying  blood-serum.     (Kocn.) 

At  the  end  of  this  time  the  serum  in  the  tubes  may 
either  be  retained  as  fluid  serum  or  solidified  at  between 


EL  O  OD-SER  UM.  123 

76°-80°  C.  In  solidifying  the  serum  the  tubes  should 
be  placed  in  an  inclined  position,  so  that  as  great  a  sur- 
face as  possible  may  be  given  to  the  serum.  The  proc- 
ess of  solidification  requires  constant  attention  if  good 
results  are  to  be  obtained — i.  e.9  if  a  translucent,  solid 
medium  is  the  result.  If  the  old,  small  form  of  appa- 
ratus be  employed  (Fig.  19),  the  solidification  can  be 
accomplished  in  a  shorter  time  than  if  the  larger 
forms  commonly  employed  are  used.  No  definite 
rule  for  the  time  that  will  be  required  can  be  laid 
down,  for  this  is  not  constant.  If  the  small  solidify- 
ing apparatus  be  used,  very  good  results  may  be  ob- 
tained in  about  two  hours  at  78°  C.  It  frequently 
requires  a  longer  time  at  a  higher  temperature  than 
has  been  mentioned.  This  is  especially  the  case  with 
Loffler's  serum-mixture. 

The  best  results  are  obtained  when  a  low  temperature 
is  employed  for  a  long  time.  Under  any  circumstances 
the  tubes  should  be  observed  from  time  to  time  through 
the  glass  door  or  cover  with  which  the  solidifying  oven 
is  provided,  and  each  time  the  oven  should  be  slightly 
jarred  with  the  hand  to  see  if  solidification,  as  indi- 
cated by  the  disappearance  of  tremors  from  the  serum, 
is  beginning.  If  the  temperature  gets  too  high,  or  the 
exposure  is  too  long,  an  opaque  medium  results.  The 
temperature  to  be  observed  is  that  of  the  air  inside 
the  chamber,  and  also  that  of  the  water  surrounding  it. 
The  latter  is  usually  a  degree  or  two  higher  than  the 
former.  The  tubes  should  not  rest  directly  upon  the 
heated  bottom  or  against  the  heated  sides  of  the  cham- 
ber, but  should  lie  upon  racks  of  wood  or  wire,  and  be 
protected  from  the  sides  by  a  wire  screen  or  gauze  :  in 
this  way  all  the  tubes  are  exposed  to  about  the  same 


124  BACTERIOLOGY. 

temperature.  The  thermometer  which  indicates  the 
temperature  inside  the  chamber  should  not  touch  the 
surfaces,  but  should  either  be  suspended  free  from 
above  through  a  cork  in  the  top  of  the  apparatus,  if 
a  large  form  of  apparatus  be  used ;  or  should  lie  upon 
a  rack  of  cork  or  wood,  its  bulb  being  free  and  a  little 
lower  than  the  other  extremity,  if  the  small,  old-fash- 
ioned apparatus  of  Koch  be  employed.  The  latter  form 
is  preferable,  as  it  is  more  easily  managed. 

When  solidification  is  complete  the  tubes  are  to  be 
retained  in  the  erect  position,  and,  unless  they  are 
intended  for  immediate  use,  must  be  prevented  from 
drying.  The  superfluous  ends  of  the  cotton  plugs 
should  be  burned  off,  and  the  mouths  of  the  tubes  may 
then  be  covered  by  sterilized  rubber  caps,  or,  as  Ghris- 
key  suggests,  they  may  be  closed  with  sterilized  corks 
pushed  in  on  top  of  the  cotton  plugs.  Even  with  the 
greatest  care  it  not  uncommonly  happens  that  one  or 
two  of  a  lot  of  tubes  thus  prepared  and  protected  will 
become  contaminated.  This  is  usually  due  to  spores  of 
moulds  that  have  fallen  into  the  rubber  caps  or  on  the 
cotton  plugs  during  manipulation,  and,  finding  no 
means  of  outward  growth,  have  thrown  their  hyphse 
downward  through  the  cotton  into  the  tube,  and  their 
spores  have  fallen  on  the  surface  of  the  serum  and 
developed  there. 

The  foregoing  is,  in  the  main,  the  plan  originally 
recommended  by  Koch  for  the  preparation  of  this 
medium.  In  recent  times,  however,  particularly  since 
the  diagnosis  of  diphtheria  by  the  method  of  Loffler  has 
become  so  general,  and  large  quantities  of  serum-tubes 
have  been  found  necessary,  modifications  have  been  sug- 
gested that  have,  in  this  country  at  least,  almost  entirely 


BLOOD-SERUM.  125 

supplanted  the  method  of  Koch.  These  modifications 
comprehend  both  the  source  and  the  manner  of  obtain- 
ing the  serum,  and  the  method  of  subsequently  steriliz- 
ing it.  In  the  first  place,  it  is  becoming  more  and  more 
the  custom  to  obtain  serum  from  horses,  not  because  it 
possesses  any  nutritive  advantages  over  that  from 
bovines,  but  because  it  can,  in  some  large  cities  at 
least,  be  easily  obtained  from  the  laboratories  in  which 
horses  are  used  in  the  production  of  antitoxins.  In 
these  places  the  blood  is  drawn  direct  from  the  jugular  or 
some  other  large  vein  by  means  of  a  trocar  thrust  through 
the  skin  into  the  vessel.  The  result  is  that  the  animal 
is  not  injured,  the  blood  is  obtained  in  a  cleanly  manner, 
and  when  due  precautions  are  taken  it  is  almost  free 
from  bacterial  contamination,  so  that  the  sterilization 
of  the  serum  obtained  from  it  offers  little  or  no  diffi- 
culty. For  particular  purposes  blood  and  serum  are 
often  obtained  in  a  somewhat  similar  manner  from  other 
animals. 

For  the  sterilization  of  serum  the  method  now  in 
vogue  is  that  of  Councilman  and  Mallory.  Its  popu- 
larity is  due  to  the  following  facts :  by  it  the  serum  is 
more  quickly  and  easily  prepared ;  rigid  precautions 
against  contamination  during  collection  of  the  serum  are 
not  so  necessary ;  and  the  resulting  medium,  while  not 
transparent  or  even  translucent  (points  aimed  at  in  the 
original  method),  fully  meets  all  the  requirements. 

The  special  points  in  the  method  are :  the  serum  is 
decanted  into  test-tubes  as  soon  as  obtained;  it  is  then 
firmly  coagulated  in  a  slanting  position  in  the  dry-air 
sterilizer  at  from  80°  to  90°  C. ;  it  is  then  sterilized  in 
the  steam  sterilizer  at  100°  C.  on  three  successive  days, 
as  in  the  case  of  other  culture-media.  It  may  then  be 


126  BACTERIOLOGY. 

protected  against  evaporation  by  sterilized  rubber  caps 
or  sterilized  corks  in  the  way  already  described,  and  set 
aside  until  needed. 

Unless  the  coagulation  in  the  dry  sterilizer  be  com- 
plete, the  surface  of  the  serum  will  be  found  to  be  blis- 
tered and  pitted  by  bubbles  and  cavities  after  it  has 
been  subjected  to  the  steam  sterilization.  A  similar 
formation  of  cavities  over  the  surface  of  the  serum  will 
occur  if  the  temperature  of  the  hot-air  sterilizer,  in 
which  it  is  solidified,  is  allowed  to  get  above  90°  C., 
or  if  it  be  elevated  to  this  point  too  quickly. 

It  is  of  no  special  advantage  to  have  the  serum  clear, 
as  the  admixture  of  blood-coloring-matter  does  not  affect 
its  nutritive  properties. 

METHODS  OF  OBTAINING  BLOOD-SERUM. — It  is  often 
desirable  to  obtain  small  quantities  of  blood-serum 
under  strictly  aseptic  precautions,  and  for  this  purpose 
Nuttall1  suggests  a  very  convenient  method.  By  the 
use  of  a  sterilized  vessel,  of  the  shape  shown  in  Fig. 
20,  from  10  to  100  c.c.  of  blood  can  be  collected,  and  if 
proper  precautions  are  observed  no  contamination  by 
bacteria  need  occur.  The  collecting  bulb  is  used  in  the 
following  way :  an  artery,  either  femoral  or  carotid,  is 
exposed,  and  around  it  two  ligatures  are  placed  ;  that 
distant  from  the  heart  is  tightened,  while  the  one  near- 
est the  heart  is  left  loose ;  between  the  latter  and  the 
heart  the  artery  is  clamped.  A  small  slit  is  then  made 
in  its  wall,  into  which  the  point  a  of  the  bulb  is  intro- 
duced and  the  artery  bound  tightly  around  it  with  the 
hitherto  loose  ligature  ;  the  clamp  is  removed  and  the 
bulb  quickly  fills  with  blood.  The  clamp  is  now  again 
put  in  position,  the  point  of  the  bulb  removed  and 

1  Nuttall :  Centralbl.  f.  Bakt.  u.  Parasitenkunde,  1892,  vol.  xi.  p.  539. 


BLOOD-SERUM.  127 

sealed  in  a  gas-flame,  the  loose  ligature  tightened, 
the  wound  closed,  and  the  bulb  containing  the  blood  is 
set  aside  in  a  cool  place  until  coagulation  has  occurred. 
The  serum  is  most  easily  withdrawn  from  the  bulb  by 
means  of  a  pipette,  closed  above  with  a  cotton-plug,  and 
supplied  with  a  piece  of  rubber-tubing  about  one-half 
metre  long,  with  glass  mouth-piece.  By  holding  the 
pipette  in  the  hand  and  sucking  upon  the  rubber  tube 
one  can  more  easily  direct  the  point  of  the  pipette  than 

FIG.  20. 


a 

Nuttall's  bulb  for  collecting  blood-serum  under  antiseptic  precautions. 

if  it  is  used  in  the  ordinary  way.  The  bulbs  are 
easily  blown,  and  after  having  been  sealed  at  the  point 
and  plugged  with  cotton  can  be  kept  on  hand  just  as 
are  sterilized  test-tubes.  An  ordinary  test-tube  drawn 
out  at  the  bottom  to  a  fine  point  may  be  substituted 
for  the  pear-shaped  bulb  with  equally  satisfactory 
results. 

Latapie  l  describes  an  apparatus  for  the  collection  of 

1  Latapie  :  Annales  dc  1'Institut  Pasteur,  1900,  vol.  xiv.  p.  106. 


128  BACTERIOLOGY. 

small  amounts  of  blood-serum  from  experimental  ani- 
mals which  has  been  found  very  useful.  The  amount 
of  serum  that  can  be  obtained  in  this  manner  from  a 
small  quantity  of  blood  is  much  greater  than  in  the 
Nuttall  bulb.  The  Latapie  apparatus  has  been  very 
much  improved  and  simplified  by  Rivas.1  The  advan- 
tages of  the  Rivas  modification  are  that  it  is  much  more 
easily  constructed  and  is  far  more  durable — that  is,  it  is 
less  liable  to  break  during  sterilization  and  in  subsequent 
manipulations. 

The  Rivas  apparatus  is  constructed  from  two  test- 
tubes  about  15  x  180  mm.  in  size.  The  mouth  of 
one  test-tube  is  drawn  out  into  a  long  narrow  neck 
1  cm.  in  diameter  and  about  5  cm.  in  length.  Three 
or  four  points  on  the  side  of  the  tube  are  softened  in 
the  flame  of  a  blowpipe,  and  the  softened  glass  driven 
inward  by  means  of  a  piece  of  pointed  wood.  This 
gives  supports  on  the  interior  of  the  tube  to  hold  the 
coagulated  blood  in  place.  Between  the  long  narrow 
neck  and  the  body  of  the  tube  a  constriction  is  formed 
by  drawing  out  the  tube  while  heated.  The  second  tube 
also  has  a  similar  constriction  about  20  cm.  from  its 
mouth. 

The  two  tubes  are  now  fitted  together  by  inserting  the 
one  with  the  long  narrow  neck  into  the  second  tube ;  a 
small  amount  of  cotton  being  first  carefully  folded 
around  the  neck  of  the  first  tube,  so  as  to  prevent  the 
entrance  of  dust.  The  two  tubes  are  then  fastened  to- 
gether by  means  of  a  wire  twisted  around  the  constric- 
tion at  the  neck  of  each  tube,  and  the  apparatus  is  then 
wrapped  in  cotton  and  sterilized  in  a  hot-air  sterilizer. 

1  Eivas:  University  of  Pennsylvania  Medical  Bulletin,  vol.  xvii. 
1901,  p.  295. 


BLOOD-SERUM. 


129 


Before  using  the  apparatus  the  extremity  of  the  first 
tube  is  heated  in  the  gas-flame,  and  by  touching  this 
point  with  a  piece  of  pointed  glass  rod  it  is  gently  drawn 


FIG.  21. 


U 


Rivas  apparatus  for  collecting  blood-serum  :  A,  long  narrow  neck  on  first 
tube ;  B,  constriction  on  tubes  near  mouth  ;  C,  invaginations  on  first  tube  ; 
D,  small  cannula  drawn  out  on  extremity  of  first  tube ;  E,  blood-clot,  and  /', 
blood-serum  collected  in  bottom  of  second  tube. 

out  into  a  fine  cannula.    When  the  animal  has  been  pre- 
pared for  the  operation  and  a  vessel  exposed,  the  point 

9 


130  BA  CTERIOL  OGY. 

of  the  cannula  is  snipped  off  with  a  sterile  scissors,  when 
the  point  of  the  cannula  is  inserted  into  the  vessel.  The 
pressure  of  blood  is  sufficient  to  iill  the  first  tube.  The 
point  of  the  cannula  is  now  removed  from  the  vessel  and 
sealed  in  a  gas-flame.  The  apparatus  is  laid  aside  in  an 
almost  horizontal  position  until  the  blood  has  become 
completely  coagulated.  It  is  then  inverted  and  set 
aside  for  the  serum  to  separate  and  trickle  down  through 
the  narrow  neck  of  the  first  tube  and  collect  in  the 
second  tube.  When  this  has  occurred,  the  wire  holding 
the  two  tubes  together  is  unwound,  and  the  first  tube  is 
removed  and  the  second  plugged  with  a  well-fitting 
sterile  cotton  plug,  when  the  serum  may  be  preserved 
in  the  tube  for  several  days  without  danger  of  con- 
tamination. 

PRESERVATION  OF  BLOOD-SERUM.  —  It  is  some- 
times desirable  to  preserve  blood-serum  in  a  fluid 
state.  This  can  be  done  by  the  fractional  method 
of  sterilization  at  low  temperatures,  already  described, 
or  with  much  less  effort,  and  without  the  use  of  heat, 
by  a  method  that  we  have  found  very  satisfactory.  In 
the  course  of  Kirschner's  investigations  chloroform  was 
shown  to  possess  decided  disinfectant  properties  ;  as  it  is 
quite  volatile,  it  is  easily  got  rid  of  when  its  disinfectant 
or  antiseptic  properties  are  no  longer  required.  If,  there- 
fore, the  serum  to  be  preserved  be  placed  in  a  closely 
stoppered  flask  and  enough  chloroform  added  to  form  a 
thin  layer,  about  2  mm.,  on  the  bottom,  the  serum  may 
be  kept  indefinitely  without  contamination,  so  long  as 
the  chloroform  is  not  permitted  to  evaporate.  This 
latter  provision  is  one  on  which  success  depends.  If 
the  vessel  containing  the  mixture  of  chloroform  and 
serum  be  not  tightly  corked,  the  chloroform  vapor 


i 


SPECIAL  MEDIA.  131 

escapes  pretty  rapidly  and  exerts  no  preservative  action. 
In  fact,  bacteria  will  grow  uninterruptedly  in  a  cotton- 
stoppered  test-tube  containing  bouillon  to  which  chloro- 
form has  been  added.  When  required  for  use,  the 
serum  is  decanted  into  test-tubes,  which  are  then  placed 
in  a  water-bath  at  about  50°  C.  until  all  the  chloro- 
form has  been  driven  off;  this  can  be  determined 
by  the  absence  of  its  characteristic  odor.  The  serum 
may  then  be  solidified,  sterilized  by  heat,  and  em- 
ployed for  culture  purposes.  We  have  found  serum 
so  preserved  to  answer  all  requirements  as  a  culture- 
medium. 

SPECIAL  MEDIA. — The  media  just  described — bou- 
illon, nutrient  gelatin,  nutrient  agar-agar,  potato,  and 
blood-serum — are  those  in  general  use  in  the  laboratory 
for  purposes  of  isolation  and  study  of  the  ordinary 
forms  of  bacteria.  For  the  finer  points  of  differentia- 
tion special  media  have  been  suggested ;  a  few  of  them 
will  be  mentioned. 

Milk.  Fresh  milk  should  be  allowed  to  stand  over 
night  in  an  ice-chest,  the  cream  then  removed,  and  the 
remainder  of  the  milk  pipetted  into  test-tubes,  about 
8  c.c.  to  each  tube,  and  sterilized  by  the  intermittent 
process,  at  the  temperature  of  steam,  for  three  succes- 
sive days. 

The  separation  of  the  cream  may  be  accelerated  and 
rendered  more  complete  if  the  cylinder  containing  the 
milk  be  placed  in  the  steam  sterilizer  for  fifteen  minutes 
before  it  is  placed  in  the  ice-chest. 

The  cream  is  best  separated  from  the  milk  by  the  use 
of  a  cylindrical  vessel  with  a  stopcock  at  the  bottom,  by 
means  of  which  the  milk,  devoid  of  cream,  may  be 
drawn  off.  A  Chevalier  creamometer  with  a  stopcock 


1 32  BA  CTERIOLOG  Y. 

at  the  bottom  serves  the  purpose  very  well.  It  should 
be  covered  while  standing.1 

Milk  may  be  used  as  a  culture-medium  without  any 
addition  to  it,  or,  before  sterilizing,  a  few  drops  of 
litmus  tincture  may  be  added,  just  enough  to  give  it  a 
pale-blue  color.  By  this  means  it  will  be  seen  that 
different  organisms  bring  about  different  reactions  in 
the  medium :  some  producing  alkalies,  which  cause  the 
blue  color  to  be  intensified ;  others  producing  acids,  which 
change  it  to  red ;  while  others  bring  about  neither  of 
these  changes.  Similarly  litmus  solution  is  often  added 
to  gelatin  and  agar-agar  for  the  same  purpose. 

Milk  may  also  be  employed  as  a  solid  culture-medium 
by  the  addition  to  it  of  gelatin  or  agar-agar  in  the  pro- 
portions given  for  the  preparation  of  ordinary  nutri- 
ent gelatin  or  agar-agar.  It  has,  however,  in  this  form 
the  disadvantage  of  not  being  transparent,  and  can 
therefore  best  be  used  for  the  study  of  those  organisms 
which  grow  upon  the  surface  of  the  medium  without 
causing  liquefaction. 

Nutrient  gelatin  and  agar-agar  can  also  be  prepared 
from  neutral  milk-whey,  obtained  from  milk  after  pre- 
cipitation of  the  casein. 

Litmus-milk-whey.  An  important  differential  medium 
is  milk-whey  to  which  litmus  tincture  has  been  added. 
A  number  of  methods  for  its  preparation  are  in  use,  but 
the  one  employed  by  Durham  seems  to  be  the  most  sat- 
isfactory. Briefly  it  is  as  follows :  fresh  milk,  free 
from  antiseptic  adulterations,  is  gently  warmed  and 
clotted  with  essence  of  rennet.  The  whey  is  strained 

1  For  some  time  past  we  have  been  using  what  is  technically  known 
as  "separator  milk  " — i.  e.,  the  fluid  left  after  milk  has  been  deprived 
of  its  fat  (cream)  by  centrifugal  force. 


7iTYJ 

SPECIAL  MEDIA.  133 

off  and  the  clot  hung  up  to  drain  in  a  piece  of  muslin. 
The  whey,  which  is  somewhat  turbid  and  yellow,  is 
then  cautiously  neutralized  with  a  4  per  cent,  citric  acid, 
solution,  neutral  litmus  solution  being  used  as  the  indi- 
cator. It  is  then  heated  upon  a  water-bath  to  100°  C. 
for  about  half  an  hour ;  thereby  nearly  the  whole  of  the 
proteid  is  coagulated.  It  is  then  filtered  clear  and  neu- 
tral litmus  solution  is  added  until  it  is  of  a  distinct  pur- 
ple color.  If  the  filtered  whey  is  cloudy,  let  it  stand  in 
a  cold  place  for  a  day  or  two  and  decant  off  the  clear 
supernatant  portion  or  pass  it  through  a  Berkfeld  filter. 
The  whey  should  never  be  heated  above  100°  C.  or 
neutralized  with  mineral  acids,  otherwise  there  is  a 
danger  of  so  modifying  the  milk-sugar  present  as  seri- 
ously to  impair  the  usefulness  of  the  medium.  When 
properly  prepared,  the  medium  is  free  from  proteid,  and 
contains  only  water,  lactose,  the  salts  of  the  milk,  and 
a  small  quantity  of  a  body  suggestive  of  dextrose  or 
galactose.  The  medium  is  of  great  utility  in  detecting 
the  power  of  bacteria  to  cause  acid  fermentation  in  a 
non-proteid  medium  containing  a  fermentable  sugar; 
and  for  observing  the  variations  of  this  power  in  closely 
allied  though  not  identical  species. 

Dunham's  peptone  solution.  The  medium  usually 
known  as  Dunham's  solution  is  prepared  according  to 
the  following  formula : 

Dried  peptone 1    part 

Sodium  chloride 0.5     " 

Distilled  water 100    parts. 

It  is  usually  of  a  neutral  or  slightly  alkaline  reac- 
tion, and  neutralization  is  not,  therefore,  necessary. 
It  is  filtered,  decanted  into  tubes  or  flasks,  and  ster- 


1 34  BA  CTERIOL  OGY. 

ilized  in  the  steam  sterilizer  in  the  ordinary  way. 
The  most  common  use  to  which  this  solution  is  put 
is  in  determining  if  the  organism  under  considera- 
tion possesses  the  property  of  producing  indol  as  one 
of  its  metabolic  products.  It  is  essential  for  accu- 
racy that  the  preparation  of  dried  peptone  employed 
should  be  as  nearly  chemically  pure  as  is  possible, 
and  indeed  the  other  ingredients  should  be  corre- 
spondingly free  from  impurities.  Gorini1  calls  attention 
to  the  fact  that  impurities  in  the  peptone,  particularly 
the  presence  of  carbohydrates,  so  interfere  with  the 
production  of  indol  by  certain  bacteria  that  otherwise 
produce  it,  that  it  is  ofttimes  impossible,  under  such  cir- 
cumstances, to  obtain  the  characteristic  color-reaction  of 
this  body,  and  where  it  is  obtained  it  is  always  after  a 
much  longer  time  than  is  the  case  where  peptone  free 
from  these  substances  has  been  used. 

Peckham  has  also  demonstrated  that  where  bacteria 
have  the  property  of  forming  indol  and  also  of  fer- 
menting carbohydrates,  their  proteolytic  function,  as 
evidenced  by  the  appearance  of  indol  as  a  product 
of  metabolism,  may  be  completely  suppressed  by  the 
addition  of  such  fermentable  carbohydrates  as  glucose, 
saccharose,  and  lactose  to  the  proteid  solution  in  which 
they  are  developing.2 

Gorini  suggests  the  advisability  of  testing  the  purity 
of  all  peptone  preparations  before  using  them,  by  means 
of  the  reaction  that  they  exhibit  with  Fehling's  alka- 
line copper  solution.  Under  the  influence  of  this  re- 
agent pure  peptone  in  solution  gives  a  violet  color  (the 

1  Gorini :  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  1893, 
vol.  xiii.  p.  790. 

2  See  Journal  of  Experimental  Medicine,  1897,  vol.  ii.  p.  549. 


SPECIAL  MEDIA.  135 

bitiret  reaction),  which  remains  permanent  even  after 
boiling  for  mve  minutes.  If,  instead  of  a  violet  color, 
there  appears  a  red  or  reddish-yellow  precipitate,  the 
peptone  should  be  discarded,  as  in  his  experience  no 
indol  is  produced  from  peptone  giving  this  reaction. 
Both  the  peptone  solution  and  that  of  the  copper  (par- 
ticularly the  latter)  should  be  relatively  dilute  in  order 
for  the  reaction  to  be  successful. 

Lactose  litmus-agar,  or  litmus-gelatin  of  Wurtz.  A 
medium  of  much  use  in  the  differentiation  of  bacteria 
is  that  recommended  by  Wurtz,  consisting  of  slightly 
alkaline  nutrient  agar-agar,  to  which  from  2  to  3  per 
cent,  of  lactose  and  sufficient  litmus  tincture  to  give 
it  a  pale-blue  color  have  been  added.  Bacteria  capable 
of  causing  fermentation  of  lactose  when  grown  on  this 
medium  develop  into  colonies  of  a  pale-pink  color  and 
cause,  likewise,  a  reddening  of  the  surrounding  medium, 
owing  to  the  production  of  acid  as  a  result  of  their 
action  upon  the  lactose  ;  while  other  bacteria,  incapable 
of  such  fermentative  activities,  grow  as  pale-blue  colonies 
and  cause  no  reddening  of  the  surrounding  medium. 
It  is  especially  useful  in  the  differentiation  of  the  bacillus 
of  typhoid  fever,  which  does  not  possess  the  property 
of  bringing  about  fermentation  of  lactose,  from  other 
organisms  that  simulate  it  in  many  other  respects,  but 
which  do  possess  this  property. 

Its  preparation  is  as  follows :  to  nutrient  agar-agar 
or  gelatin,  the  alkalinity  of  which  is  such  that  1  c.c. 
\\ill  require  0.1  c.c.  of  a  1  :  20  normal  sulphuric-acid 
solution  to  neutralize  it,  lactose  is  added  in  the  propor- 
tion of  2  or  3  per  cent. ;  it  is  then  decanted  into  test- 
tubes  and  sterilized  in  the  usual  way.  When  ster- 
ili/ation  is  complete  enough  sterilized  litmus  tincture 


136  BACTERIOLOGY. 

should  be  added  to  each  tube  to  give  a  decided,  though 
not  very  intense,  blue  color.  This  must  be  done  care- 
fully, to  avoid  contamination  of  the  tubes  during  ma- 
nipulation. It  is  better  not  to  add  the  litmus  tincture 
before  sterilizing  the  tubes,  as  its  color-characteristics 
are  altered  by  contact  with  organic  matters  under  the 
influence  of  heat.  This  medium  is  used  for  both  test- 
tube  and  plate  cultivation,  just  as  is  ordinary  agar-agar 
and  gelatin. 

Loffler's  blood-serum  mixture.  Loffler's  blood-serum 
mixture  consists  of  one  part  of  neutral  meat-infusion 
bouillon,  containing  1  per  cent,  of  grape-sugar,  and 
three  parts  of  blood-serum.  This  mixture  is  placed  in 
test-tubes,  sterilized,  and  solidified  in  exactly  the  way 
given  for  blood-serum.  It  requires  for  its  solidification 
a  somewhat  higher  temperature  and  a  longer  exposure 
to  this  temperature  than  does  blood-serum  to  which  no 
bouillon  has  been  added.  (See  also  the  Councilman- 
Mallory  method.) 

The  Serum-water  Media  of  Hiss. — A  medium  which 
has  been  found  very  serviceable  in  the  differentiation 
between  closely  related  bacteria  is  prepared  by  mixing 
one  part  of  blood-serum  (either  horse  or  bovine)  and 
three  parts  of  distilled  water.  This  is  neutralized,  and 
heated  in  a  water-bath  or  an  Arnold  steam  sterilizer 
until  it  becomes  opalescent.  A  5  per  cent,  aqueous 
solution  of  litmus  is  then  added  in  the  proportion  of  1 
per  cent.  Any  one  of  the  carbohydrates,  as  dextrose, 
lactose,  saccharose,  levulose,  mannite,  etc.,  is  then  added 
in  the  proportion  of  1  per  cent.  The  finished  medium 
is  then  placed  in  test-tubes.  The  medium  must  be  ster- 
ilized in  an  Arnold  steam  sterilizer,  and  it  is  advisable 
to  allow  the  sterilizer  to  remain  uncovered  during  the 


SPECIAL  MEDIA.  137 

process  of  sterilization  to  avoid  excessive  heating  of  the 
medium. 

The  relative  degree  of  acidity  produced,  with  or  with- 
out coagulation,  with  or  without  gas-production,  and 
with  or  without  reduction  of  the  litmus,  in  a  series  of 
tubes  of  this  medium  containing  the  different  carbohy- 
drates serves  to  differentiate  between  related  species  of 
bacteria.  For  instance,  the  colon  bacillus  produces  an 
acid  reaction  with  coagulation  and  gas-formation  with 
some  of  the  carbohydrates,  while  the  typhoid  bacillus 
produces  a  lower  degree  of  acidity  with  coagulation,  but 
without  gas-production.  Similarly,  the  different  types 
of  the  dysentery  bacillus  may  be  differentiated  by  means 
of  their  effects  on  the  different  carbohydrates  in  this 
medium. 

A  complete  list  of  the  special  media  would  be  too 
voluminous  for  a  book  of  this  size.  For  their  descrip- 
tion the  reader  is  referred  to  the  current  literature. 
Those  that  have  been  given  above  will  suffice  for  ob- 
taining a  clear  understanding  of  the  principles  of  the 
subject.  In  the  chapters  upon  the  Pathogenic  Bacteria 
such  special  media  as  have  proved  of  use  for  purposes 
of  identification  and  differentiation  are  described  in 
detail. 


CHAPTER    VI. 

Preparation  of  the  tubes,  flasks,  etc.,  in  which  the  media  are  to  be 
preserved. 

. 

WHILE  the  media  are  in  course  of  preparation  it  is 
well  to  get  the  test-tubes  and  flasks  ready  for  their 
reception,  and  it  is  essential  that  they  should  be  as  clean 
as  it  is  possible  to  make  them.  For  this  purpose  it  is 
advisable  that  both  new  tubes  and  those  which  have 
previously  been  used  should  be  boiled  for  about  thirty 
to  forty-five  minutes  in  a  2  to  3  per  cent,  solution  of 
common  soda ;  it  is  not  necessary  to  be  exact  as  to 
strength,  but  it  should  not  be  weaker  than  this.  At  the 
end  of  this  time  they  are  to  be  carefully  swabbed  out 
with  a  cylindrical  bristle  brush,  preferably  one  with 
a  reed  handle  (Fig.  22,  a),  as  those  with  wire  handles 

FIG.  22. 
a 


Brushes  for  cleaning  test-tubes. 


are  apt  to  break  through  the  bottoms  of  the  tubes, 
though  Messrs.  Lentz  &  Sons,  of  this  city,  have  in 
large  part  eliminated  this  objection  from  the  wire-handle 
brush  depicted  in  Fig.  22,  6.  All  traces  of  adherent 


138 


FILLING   THE  TUBES.  139 

material  should  be  carefully  removed.  When  the  tubes 
are  quite  clean  they  may  be  rinsed  in  a  warm  solution 
of  commercial  hydrochloric  acid  of  the  strength  of 
about  1  per  cent.  This  is  to  remove  the  alkali.  They 
are  then  to  be  thoroughly  rinsed  in  clear,  running  water, 
and  stood  top  down  until  the  water  has  drained  from 
them.  When  dry  they  are  to  be  plugged  with  raw 
cotton ;  this  requires  a  little  practice  before  it  can  be 
properly  done.  The  cotton  should  be  introduced  into 
the  mouths  of  the  tubes  in  such  a  way  that  no  cracks 
or  creases  exist.  The  plug  should  fit  neither  too  tightly 
nor  too  loosely,  but  should  be  just  firmly  enough  in 
position  to  sustain  the  weight  of  the  tube  into  which 
it  is  placed  when  held  up  by  the  portion  which  projects 
from  and  overhangs  the  mouth  of  the  tube.  The  tubes 
thus  plugged  are  now  to  be  placed  upright  in  a  wire 
basket  and  heated  for  one  hour  in  the  hot-air  sterilizer 
at  a  temperature  of  about  150°  C.  A  very  good  guide 
for  this  process  of  sterilization  is  to  observe  the  tubes 
from  time  to  time,  and  as  soon  as  the  cotton  has  become 
a  very  light-brown  color,  not  deeper  than  a  dark-cream 
tint,  to  consider  sterilization  complete.  The  tubes  are 
then  removed  and  allowed  to  cool. 

The  cotton  used  for  this  purpose  should  be  the  ordi- 
nary cotton  batting  of  the  shops,  and  not  absorbent 
cotton ;  the  latter  becomes  too  tightly  packed,  and  is, 
moreover,  much  too  expensive  for  this  purpose. 

Care  should  be  taken  not  to  burn  the  cotton,  other- 
wise the  tubes  will  become  coated  with  a  dark-colored, 
empyreumatic,  oily  deposit,  which  necessitates  recleans- 
ing. 

FILLING  THE  TUBES. — When  the  tubes  are  cold 
they  may  be  filled.  This  is  best  accomplished  by  the 


140 


BACTERIOLOGY. 


use  of  a  separating  funnel,  such  as  is  shown  in  Fig. 
23.  The  liquefied  medium  is  poured  into  this  funnel, 
which  has  been  carefully  washed,  and  by  pressing  the 
pinchcock  with  which  the  funnel  is  provided  the  desired 
amount  of  material  (5-10  c.c.)  may  be  allowed  to  flow 
into  the  tubes  held  under  its  opening.  It  is  not  neces- 
sary to  sterilize  the  funnel,  for  the  medium  is  to  be  sub- 
jected to  this  process  as  soon  as  it  is  in  the  test-tubes. 

FIG.  23. 


Funnel  for  filling  tubes  with  culture-media. 

Care  should  be  taken  that  none  of  the  medium  is 
dropped  upon  the  mouth  of  the  test-tube,  otherwise  the 
cotton  plug  becomes  adherent  to  it,  and  is  not  only 
difficult  to  remove,  but  presents  a  very  untidy  appear- 
ance and  interferes  materially  with  the  manipulations. 


FILLING   THE  TUBES.  141 

As  soon  as  the  tubes  have  been  filled  they  are  to  be 
sterilized  in  the  steam  sterilizer  for  fifteen  minutes  on 
each  of  three  successive  days.  During  the  intervening 
days  they  may  be  kept  at  the  ordinary  room-tempera- 
ture. 

When  sterilization  is  complete  and  the  medium  in 
the  tubes  is  still  liquid,  some  of  them  may  be  placed  in 
a  slanting  position,  at  an  angle  of  about  ten  degrees 
with  the  surface  on  which  they  rest,  and  the  medium 
allowed  to  solidify  in  this  position.  These  are  for  the 
so-called  slant-cultures.  The  remainder  may  solidify  in 
the  erect  position ;  these  serve  for  making  plates. 

For  Esmarch  tubes  not  more  than  5  c.c.  of  material 
should  be  placed  in  each  tube,  as  more  than  this  renders 
it  difficult  to  distribute  the  gelatin  evenly  over  the  inner 
surface  of  the  tubes  when  they  are  rolled. 


CHAPTER  VII. 

Technique  of  making  plates— Petri  plates.  Esmarch  tubes,  etc. 

PLATES. — The  plate  method  can  be  employed  with 
both  agar-agar  and  gelatin.  It  cannot  be  practised  with 
blood-serum,  because  the  serum  when  once  solidified 
cannot  be  again  liquefied. 

Plates  are  usually  referred  to  as  "  a  set."  This 
term  implies  three  individual  plates,  each  representing 
a  mixture  of  organisms  in  a  higher  state  of  dilution. 
The  first  plate  is  known  usually  as  "  the  original/7  or 
" plate  No.  1,"  the  first  dilution  from  this  as  "plate 
No.  2,"  and  the  second  as  "  plate  No.  3." 

In  the  preparation  of  a  set  of  plates  the  following 
are  the  steps  to  be  observed  : 

Three  tubes,  each  containing  from  7  to  9  c.c.  of  gela- 
tin or  agar-agar,  are  placed  in  a  warm  water-bath 
until  the  medium  has  become  liquid.  If  agar-agar  is 
employed,  this  is  accomplished  at  the  boiling-point  of 
water;  if  gelatin  is  used,  a  much  lower  temperature 
suffices  (35°-40°  C.).  When  liquefaction  is  complete 
the  temperature  of  the  water,  in  the  case  of  agar-agar, 
must  be  reduced  to  41°-42°  C.,  at  which  temperature  ; 
the  agar-agar  remains  liquid,  and  the  organisms  may  . 
be  introduced  into  it  without  fear  of  destroying  their 
vitality.  The  medium  being  now  liquid  and  of  the 
proper  temperature,  a  very  small  portion  of  the  mixture 
of  organisms  to  be  studied  is  taken  up  with  a  sterilized 

142 


TECHNIQUE  OF  MAKING  PLATES.  143 

platinum  wire  (Fig.  24,  a)  about  5  cm.  long,  twisted 
into  a  small  loop  at  one  end  and  fused  into  a  bit 
of  glass  rod,  which  serves  as  a  handle,  at  the  other 
extremity.  This  loop  is  one  of  the  most  useful  of  bac- 
teriological instruments,  as  there  is  hardly  a  manipula- 
tion into  which  it  does  not  enter.  Under  no  circum- 
stances is  it  to  be  employed  without  having  been 
passed  through  a  gas-flame  until  quite  hot,  for  the 
purpose  of  sterilization.  One  should  form  a  habit 


FIG.  24. 
a 


6 
Looped  and  straight  platinum  wires  in  glass  handles. 

of  never  taking  up  one  of  these  platinum-wire  needles, 
as  they  are  called,  for  they  are  curved  and  straight  (Fig. 
24,  6)  as  well  as  looped,  without  passing  it  through 
a  flame ;  and  the  sooner  the  beginner  learns  to  do  this 
as  a  reflex  action,  the  sooner  does  he  eliminate  one 
of  the  possible  sources  of  error  in  his  work.  It  must 
be  remembered,  though,  that  it  should  not  be  used  when 
hot,  otherwise  the  organisms  taken  upon  it  will  be  killed 
by  the  high  temperature ;  after  sterilization  in  the  flame 
one  waits  for  a  few  seconds  until  it  is  cool  before  using. 
A  minute  portion  of  the  material  under  consideration 
is  transferred  with  the  sterilized  loop  into  tube  No.  1, 
"  the  original,"  where  it  is  thoroughly  disintegrated  by 
gently  rubbing  it  against  the  sides  of  the  tuj^.  *  The 
more  carefully  this  is  done  the  more  uniform Avill  be 


144  BACTERIOLOGY. 

the  distribution  of  the  organisms  and  the  better  the 
results.  The  loop  is  then  again  sterilized  and  three 
of  its  loopfuls  are  passed,  without  touching  the  sides  of 
the  tube,  from  "  the  original "  into  tube  No.  2,  where 
they  are  carefully  mixed.  Again  the  loop  is  sterilized, 
and  again  three  dips  are  made  from  tube  No.  2  into 
tube  No.  3.  This  completes  the  dilution.  The  loop  is 
now  sterilized  before  laying  it  aside. 

FIG.  25. 


Levelling-tripod  with  glass  chamber  for  plates. 

During  this  manipulation,  which  must  be  done 
quickly  if  agar-agar  be  employed,  the  temperature  of 
the  water  in  the  bath  in  which  the  tubes  stand  should 
never  be  lower  than  39°  C.,  and  never  higher  than 
43°  C.  If  it  falls  below  38°  C.,  the  agar-agar  solid- 
ifies, and  can  only  be  redissolved  at  a  temperature 
that  would  be  destructive  to  the  organisms  which  may 
have  been  introduced  into  the  tubes.  This  is  not  of 
so  much  moment  with  gelatin,  since  it  may  readily  be 


TECHNIQUE  OF  MAKING  PLATES.  145 

redissolved  at  a  temperature  not  detrimental  to  the 
organisms  with  which  the  tubes  may  have  been  inocu- 
lated. 

THE  COOLING-STAGE  AND  LEVELLING-TRIPOD. — - 
While  the  medium  of  which  the  plates  are  to  be  made 
is  melting  it  is  well  to  arrange  the  cooling-stage  (Fig. 
25)  upon  which  the  gelatin  or  agar-agar  is  to  be  subse- 
quently solidified. 

This  stage  consists  of  a  glass  dish  filled  with  ice- 
water  and  covered  with  a  ground-glass  plate,  which  in 
turn  has  a  dome-shaped  cover.  The  dish  rests  upon  a 
tripod  which  can  be  brought  to  an  exact  level,  as  indi- 
cated by  the  spirit-level,  by  raising  or  lowering  its  legs 
by  means  of  thumb-screws,  with  which  they  are  pro- 
vided. Three  stages  are  usually  employed.  When 
ready  for  use  they  should  be  exactly  level. 

THE  GLASS  PLATES. — In  the  original  plate  method 
devised  by  Koch  the  contents  of  each  of  the  tubes  of 
agar-agar  or  gelatin  are  poured  out  in  a  thin  layer  upon 
the  surface  of  a  sterile  glass  plate.  As  soon  as  pre- 
pared, the  glass  plates  containing  the  culture  are  placed 
in  a  large  glass  culture  dish. 

PETRI'S  MODIFICATION  OF  THE  PLATE  METHOD. — 
The  modification,  now  in  general  use,  that  approaches 
nearest  to  the  original  method,  and  at  the  same  time 
lessens  very  materially  the  number  of  steps  in  the  proc- 
ess, is  that  suggested  by  Petri.  It  consists  in  substitut- 
ing for  the  plates  small,  round,  double  glass  dishes, 
having  about  the  same  surface-area  as  the  plates  (Fig. 
26).  The  liquid  medium  is  poured  directly  into  these 
little  dishes  and  their  covers  replaced  ;  they  are  then 
set  aside  for  observation.  In  all  other  respects  the 
process  is  the  same  as  Koch's  original  method.  Petri's 
10 


146  BACTERIOLOGY. 

dishes  are  about  8  cm.  in  diameter  and  about  1.5  to  2 
cm.  in  height,  the  sides  being  vertical.  They  may 
readily  be  sterilized  either  by  hot  air  or  steam.  They 
are  very  useful  for  this  work,  as  they  do  away  with  the 
necessity  for  the  cooling-stage  and  levelling-tripod, 
though  in  warm  weather  the  cooling-stage  may  be  used 
to  hasten  the  solidification  of  gelatin.  A  cooling-stage 
of  very  convenient  design  for  use  with  these  dishes 
consists  of  a  closed,  flat  metal  box,  either  of  copper  or 
block  tin,  and  round  or  square  in  shape,  so  arranged 
that  it  can  be  filled  with  cold  water,  or  that  cold  water 
can  constantly  be  passed  through  it  by  means  of  a 
rubber  tube  connected  with  a  spigot.  The  inlet  for  the 


FIG.  26. 


Petri  double  dish,  now  generally  used  instead  of  plates. 

water  should  be  just  above  the  bottom  of  the  box,  and 
the  outlet  just  beneath  the  top  and  slightly  turned 
upward  and  then  downward,  so  as  to  insure  filling  the 
space  with  water.  The  box  should  be  sufficiently  strong 
to  resist  the  pressure  of  the  water.  A  convenient  size- 
is  from  20-25  cm.  in  diameter  and  about  1.5  to  2  cm. 
high.  It  is  simple  in  construction,  and  can  be  made  by 
any  copper-spinner.  An  idea  of  its  construction  is 
given  in  Fig.  27. 

When    gelatin    or    agar-agar    is    to    be    cooled,   it 
is   only    necessary   to    place   the   dishes    containing   il 


ESMARCH  TUBES.  147 

on  top  of  this   box   and  keep  cold   water  circulating 
through  it. 

ESMAKCH  TUBES. — The  modification  of  Koch's 
method  which  insures  the  greatest  security  from  con- 
tamination by  extraneous  organisms  and  requires  the 
least  amount  of  apparatus  is  that  suggested  by  v. 
Esmarch.  It  differs  from  the  other  methods  thus  :  the 
dilutions  having  been  prepared  in  tubes  containing  a 
smaller  amount  of  medium  than  usual — as  a  rule,  not 
more  than  5  to  6  c.c. — are,  instead  of  being  poured  upon 
plates  or  into  dishes,  spread  over  the  inner  surface  of 

FIG.  27. 


Metal  cooling-stage. 

the  tubes  containing  them,  and,  without  removing  the 
cotton  plugs,  solidified  in  this  position.  The  tubes  then 
present  a  thin  cylindrical  lining  of  gelatin  or  agar-agar, 
upon  which  the  colonies  develop.  In  all  other  respects 
the  conditions  for  the  growth  of  the  organisms  are  the 
same  as  in  flat  plates. 

Esmarch  directs  that  after  completion  of  the  dilu- 
tions the  tops  of  the  cotton  plugs  in  the  test-tubes 
should  be  cut  off  flush  with  the  mouths  of  the  tubes  and 
sterilized  rubber  caps  be  placed  over  them.  They  are 


148 


BACTERIOLOGY. 


then  to  be  held  in  a  horizontal  position  and  twisted 
between  the  fingers  upon  their  long  axis  under  ice- 
water.  The  gelatin  becomes  solidified  thereby  and 
adheres  to  the  sides  of  the  tube.  When  the  gelatin  is 
quite  hard  the  tubes  are  removed  from  the  water,  wiped 
dry,  the  rubber  caps  removed,  and  the  tubes  set  aside 
for  observation. 

For  some  time  past  we  have  deviated  from  the  direc- 
tion given  by  v.  Esmarch  for  this  part  of  his  method, 
and  instead  of  rolling  the  tubes  under  ice-water,  we  roll 
them  upon  a  block  of  ice  (Fig.  28),  after  the  method 

FIG.  28. 


Demonstrating  Booker's  method  of  rolling  Esmarch  tubes  on  a  block  of  ice. 

devised  by  Booker  in  1887  in  the  Pathological  Labora- 
tory of  the  Johns  Hopkins  University.  In  this  method 
a  small  block  of  ice  only  is  needed.  It  is  levelled 
and  held  in  position  by  being  placed  upon  a  towel  in 
a  dish.  A  horizontal  groove  is  melted  in  the  upper 
surface  of  the  ice  with  a  test-tube  of  hot  water.  The 
tubes  to  be  rolled  are  then  held  in  an  almost — not  quite 


ESMARCH  TUBES.  149 

—horizontal  position  and  twisted  between  the  fingers 
until  the  sides  are  moistened  by  the  contents  to  within 
about  1  cm.  of  the  cotton  plug,  care  being  taken  that 
the  gelatin  does  not  touch  the  cotton ;  otherwise  the  latter 
becomes  adherent  to  the  sides  of  the  tube  and  is  difficult 
to  remove.  The  tube  is  then  placed  in  the  groove  in 
the  ice  and  rolled,  neither  rubber  cap  nor  cutting  off  of 
the  cotton  plug  being  necessary. 

The  advantages  of  this  process  over  that  followed  by 
v.  Esmarch  are  that  it  requires  less  time,  is  cleaner, 
no  rubber  caps  are  needed,  the  rolled  tubes  are  more 
uniform,  and  the  gelatin  does  not  touch  the  cotton  plug, 
as  is  always  the  case  in  tubes  rolled  under  water,  be- 
cause of  the  impossibility  of  keeping  them  at  one  level. 

There  is  an  erroneous  impression  that  Esmarch  tubes 
are  not  a  success  when  made  from  ordinary  nutrient 
agar-agar  because  of  the  tendency  of  this  medium  to 
shrink  and  slip  to  the  bottom  of  the  tube.  This  slip- 
ping clown  of  the  agar-agar  is  due  to  the  water,  which  is 
squeezed  from  it  during  solidification,  getting  between 
the  medium  and  the  walls  of  the  tube.  This  can  easily 
be  overcome  by  allowing  the  rolled  tubes  to  remain 
in  a  nearly  horizontal  position  for  twenty-four  hours 
after  rolling  them,  the  mouth  of  the  tube  being  about 
1  cm.  higher  than  the  bottom.  During  this  time  the 
margin  of  the  agar-agar  nearest  the  cotton  plug  dries 
and  becomes  adherent  to  the  walls  of  the  tube,  while 
the  water  collects  at  the  most  dependent  point — i.  <?.,  the 
bottom  of  the  tubes.  After  this  they  may  be  retained 
in  the  upright  position  without  danger  of  the  agar- 
agar  slipping  down.  In  all  these  manipulations, 
if  the  dilutions  of  the  number  of  organisms  have  been 
properly  conducted,  the  results  will  be  the  same.  The 


1 50  BA  CTERIOL  OGY. 

original  plate  or  tube,  as  a  rule,  will  be  of  no  use  be- 
cause of  the  great  number  of  colonies  in  it ;  plate  or 
tube  No.  2  may  be  of  service ;  but  plate  or  tube  No.  3 
will  usually  contain  the  organisms  in  such  small  num- 
bers that  there  will  be  nothing  to  prevent  the  charac- 
teristic development  of  the  colonies  originating  from 
them. 

For  reasons  of  economy  the  "  original/'  tube  No.  1,  is 
sometimes  substituted  by  a  tube  containing  normal  salt- 
solution  (0.6  to  0.7  per  cent,  of  sodium  chloride  in 
water),  which  is  thrown  aside  as  soon  as  the  dilutions 
are  completed,  and  only  plates  or  tubes  Nos.  2  and  3 
are  made. 

THE  SEKIAL  TUBE  METHOD  OP  SEPARATION. — 
Another  method  for  the  separation  of  bacteria  and 
their  isolation  as  single  colonies  consists  in  the  making 
of  dilutions  upon  the  surface  of  solid  media,  such  as 
potato,  coagulated  blood-serum,  agar-agar,  and  gelatin. 
In  pursuance  of  this  method  one  selects  a  number  of 
tubes  containing  the  medium  set  in  a  slanting  position. 
With  a  platinum  needle  a  bit  of  the  substance  to  be 
studied  is  smeared  upon  tube  No.  1  ;  without  sterilizing 
the  needle  it  is  passed  in  succession  over  the  surface 
of  the  medium  in  tubes  Nos.  2,  3,  4,  etc.  When  de- 
velopment has  occurred  essentially  the  same  conditions 
as  regards  separation  of  the  colonies  will  be  found  as 
when  plates  are  poured.  If  a  slanted  medium  be  em- 
ployed, about  the  most  dependent  angle  of  which  water 
of  condensation  has  accumulated,  as  blood-serum,  agar- 
agar,  and  potato,  the  dilutions  may  be  made  in  this 
fluid,  and  this  is  then  to  be  carefully  smeared  over  the 
solid  surface  of  the  medium.  The  tubes  thus  treated 


ESMARGH   TUBES.  151 

.should  be  kept  in  an  upright  position  to  prevent  the 
fluid  flowing  over  the  surface.  When  sufficiently  de- 
veloped, single  colonies  may  be  isolated  with  compara- 
tive ease  from  tubes  prepared  in  this  manner.  (See  also 
method  for  the  isolation  of  bacillus  diphtherice  on  blood- 
serum.) 


CHAPTER    VIII. 

The  incubating-oven — Gas-pressure  regulator — Thermo-regulator — The 
safety  burner  employed  in  heating  the  incubator. 

THE  INCUBATOR. — When  the  plates  have  been  made 
it  must  be  borne  in  mind  that  for  the  development  of 
certain  forms  of  bacteria  a  higher  temperature  is  neces- 
sary than  for  the  growth  of  others.  The  pathogenic 
or  disease-producing  organisms  grow  more  luxuriantly 
at  the  temperature  of  the  human  body  (37.5°  C.)  than 
at  lower  temperatures  ;  whereas  for  the  ordinary  sap- 
rophytic  forms  almost  any  temperature  between  18° 
and  37°  C.  is  suitable.  It  therefore  becomes  neces- 
sary to  provide  a  place  in  which  a  constant  tem- 
perature favorable  to  the  growth  of  the  pathogenic 
organisms  can  be  maintained.  For  this  purpose  a  num- 
ber of  different  forms  of  apparatus  have  been  devised. 
They  are  all  based  upon  the  same  principles,  however, 
and  a  general  description  of  the  essential  points  involved 
in  their  construction  will  be  all  that  is  needed  here. 

The  apparatus  known  as  thermostat,  incubator,  or 
brooding-oven,  is  a  copper  chamber  (Fig.  29)  with 
double  walls,  the  space  between  which  is  filled  with 
water.  The  incubating-chamber  has  a  closely  fitting 
double  door,  inside  of  which  is  usually  a  door  of  glass 
through  which  the  contents  of  the  chamber  may  be  in- 
spected without  actually  opening  it.  The  whole  appa- 
ratus is  encased  in  either  asbestos-boards  or  thick  felt, 

152 


THE  INCUBATOR. 


153 


to  prevent  radiation  of  heat  and  consequent  fluctuations 
in  temperature.  In  the  top  of  the  chamber  is  a  small 
opening  through  which  a  thermometer  projects  into  its 
interior.  At  either  corner,  leading  into  the  space  con- 
taining the  water,  are  other  openings  for  the  reception 

FIG.  29. 


Incubator  used  in  bacteriological  work. 

of  another  thermometer  and  a  thermo-regulator,  and  for 
refilling  the  apparatus  as  the  water  evaporates.  On  the 
side  is  a  water-gauge  for  showing  the  level  of  the  water 


CHAPTER    VIII. 

The  incubating-oven — Gas-pressure  regulator — Thermo-regulator — The 
safety  burner  employed  in  heating  the  incubator. 

THE  INCUBATOR. — When  the  plates  have  been  made 
it  must  be  borne  in  mind  that  for  the  development  of 
certain  forms  of  bacteria  a  higher  temperature  is  neces- 
sary than  for  the  growth  of  others.  The  pathogenic 
or  disease-producing  organisms  grow  more  luxuriantly 
at  the  temperature  of  the  human  body  (37.5°  C.)  than 
at  lower  temperatures  ;  whereas  for  the  ordinary  sap- 
rophytic  forms  almost  any  temperature  between  18° 
and  37°  C.  is  suitable.  It  therefore  becomes  neces- 
sary to  provide  a  place  in  which  a  constant  tem- 
perature favorable  to  the  growth  of  the  pathogenic 
organisms  can  be  maintained.  For  this  purpose  a  num- 
ber of  different  forms  of  apparatus  have  been  devised. 
They  are  all  based  upon  the  same  principles,  however, 
and  a  general  description  of  the  essential  points  involved 
in  their  construction  will  be  all  that  is  needed  here. 

The  apparatus  known  as  thermostat,  incubator,  or 
brooding-oven,  is  a  copper  chamber  (Fig.  29)  with 
double  walls,  the  space  between  which  is  filled  with 
water.  The  incubating-chamber  has  a  closely  fitting 
double  door,  inside  of  which  is  usually  a  door  of  glass 
through  which  the  contents  of  the  chamber  may  be  in- 
spected without  actually  opening  it.  The  whole  appa- 
ratus is  encased  in  either  asbestos-boards  or  thick  felt, 

152 


THE  INCUBATOR. 


153 


to  prevent  radiation  of  heat  and  consequent  fluctuations 
in  temperature.  In  the  top  of  the  chamber  is  a  small 
opening  through  which  a  thermometer  projects  into  its 
interior.  At  either  corner,  leading  into  the  space  con- 
taining the  water,  are  other  openings  for  the  reception 

FIG.  29. 


Incubator  used  in  bacteriological  work. 

of  another  thermometer  and  a  thermo-regulator,  and  for 
refilling  the  apparatus  as  the  water  evaporates.  On  the 
side  is  a  water-gauge  for  showing  the  level  of  the  water 


154 


BACTERIOLOGY. 


between  the  walls.  The  object  of  the  water-chamber, 
which  is  formed  by  the  double-wall  arrangement,  is  to 
insure,  by  means  of  the  warmed  water,  an  equable  tem- 
perature in  all  parts  of  the  apparatus — at  the  top  as  well 
as  at  the  sides,  back,  and  bottom  ;  the  apparatus  should 
be  kept  filled  with  water,  otherwise  the  purpose  for 
which  it  is  constructed  will  not  be  accomplished.  When 
the  chamber  between  the  walls  is  filled  with  water  heat 
is  supplied  by  a  gas-flame  placed  beneath  it. 

FIG.  30. 


Koch's  safety  burner. 


The  burner  employed  in  heating  the  incubator  was 
originally  devised  by  Koch,  and  is  known  as  "  Koch's 
safety  burner "  (Fig.  30).  It  is  a  Bunsen  burner  pro- 
vided with  an  arrangement  for  automatically  turning 
off  the  gas-supply  and  thus  preventing  accidents  should 


THERMO-REG  ULA  TORS.  1 55 

the  flame  become  extinguished  at  a  time  when  no  one 
in  near.  The  gas-cock  by  which  the  gas  is  turned  on 
and  off  is  provided  with  a  long  arm  which  is  weighted, 
and  which,  when  the  gas  is  turned  on  and  burning,  rests 
upon  an  arm  attached  to  the  side  of  a  revolving,  hori- 
zontal disk  that  is  connected  with  the  free  ends  of  two 
metal  spirals  which  are  fixed  by  their  other  ends  in  oppo- 
site directions  on  either  side  of  the  flame  and  heated  by 
it.  If  by  draughts  or  any  other  accident  the  flame  be- 
comes extinguished,  the  metal  spirals  cool,  and  in  cool- 
ing contract,  twist  the  horizontal  disk  in  the  opposite 
direction,  and  allow  the  weighted  arm  of  the  gas-cock 
to  fall.  By  its  falling  the  gas-supply  is  turned  off. 

THERMO-REGULATORS. — The  regulation  and  main- 
tenance of  the  proper  temperature  within  the  incubator 
are  accomplished  by  the  employment  of  an  automatic 
thermo-regulator. 

The  common  form  of  thermo-regulator  used  for  this 
purpose  is  constructed  upon  principles  involving  the 
expansion  and  contraction  of  fluids  under  the  influence 
of  heat  and  cold.  By  means  of  this  expansion  and  con- 
traction the  amount  of  gas  passing  from  the  source  of 
supply  to  the  burner  may  be  either  diminished  or  in- 
creased as  the  temperature  of  the  substance  in  which 
the  regulator  is  placed  either  rises  or  falls. 

The  simplest  form  of  thermo-regulator  which  serves 
to  illustrate  the  principles  involved  is- seen  in  Fig.  31. 
It  consists  of  a  glass  cylinder,  e,  having  a  communi- 
cating branch  tube  b,  and  rubber  stopper  /,  through 
which  projects  the  bent  tube  a.  The  tube  a  is  ground 
to  a  slanting  point  at  the  extremity  which  projects  into 
the  tube  e,  and  is  provided  a  short  distance  above  this 
point  with  a  capillary  opening,  g,  in  one  of  its  sides. 


158 


BACTERIOLOGY. 


as  ether,  alcohol,  and  calcium  chloride  solution,  which 
expand  and  contract  rapidly  and  regularly  under  slight 
variations  in  temperature,  are  commonly  employed ; 
whereas  for  temperatures  approaching  the  boiling-point 
of  water  mercury  is  most  frequently  used. 

The  temperature  of  the  incubator  is  to  be  regulated, 
then,  by  the  use  of  some  such  form  of  apparatus  as  that 
just  described.  It  should  be  of  sufficient  delicacy  to 
prevent  a  fluctuation  of  more  than  0.2°  C.  in  the  tem- 
perature of  the  air  within  the  chamber  of  the  apparatus. 

GAS-PRESSURE  REGULATORS. — A  gas-pressure  reg- 
ulator is  not  rarely  intervened  between  the  gas-supply 

FIG.  32. 


Moitessier's  gas-pressure  regulator. 


and  the  thermo-regulator.  This  apparatus  has  for  its 
object  the  maintenance  of  a  constant  pressure  of  the 
gas  going  to  the  thermo-regulator.  There  are  several 


GAS-PRESSURE  REGULATORS.  159 

forms  of  regulator  in  use,  but  they  do  not  accomplish 
the  object  for  which  they  are  designed. 

The  instrument  most  commonly  employed,  the  appa- 
ratus of  Moitessier  (Fig.  32),  is  based  on  somewhat  the 
same  principles  as  the  large  regulators  seen  at  the  manu- 
factories of  illuminating-gas.  Such  apparatus  act  very 
well  when  employed  on  the  large  scale,  as  one  sees  them 
at  the  gas-works ;  but  when  applied  to  the  limited  and 
sudden  fluctuations  seen  in  the  gas  coming  from  an 
ordinary  gas-cock  are  practically  useless.  They  are  too 
gross  in  their  construction,  and  act  only  under  compar- 
atively great  and  gradual  fluctuations  in  pressure.  If 
a  good  form  of  thermo-regulator  be  employed,  there  is 
no  necessity  for  the  use  of  any  of  the  pressure-regulators 
thus  far  introduced. 


CHAPTER  IX. 

The  study  of  colouies — Their  naked-eye  peculiarities  and  their  appear- 
ance under  different  conditions — Differences  in  the  structure  of 
colonies  from  different  species  of  bacteria — Stab-cultures — Slant- 
cultures. 

THE  plates  of  agar-agar  which  have  been  prepared 
from  a  mixture  of  organisms  and  have  been  placed  in 
the  incubator,  and  those  of  gelatin  which  have  been 
maintained  at  the  ordinary  temperature  of  the  room, 
are  usually  ready  for  examination  after  from  twenty-four 
to  forty-eight  hours.  They  will  be  found  marked  here 
and  there  by  small  points  or  little  islands  of  more  or 
less  opaque  appearance.  In  some  instances  these  will 
be  so  transparent  that  it  is  with  difficulty  one  can  see 
them  with  the  naked  eye.  Again,  they  may  be  of  m 
dense,  opaque  appearance ;  at  one  time  sharply  circum- 
scribed and  round,  again  irregular  in  their  outline  ;  here 
a  point  will  present  one  color,  there  perhaps  another. 
On  gelatin  some  of  the  points  will  be  seen  to  be  lying 
on  the  surface  of  the  medium,  others  will  have  sunk 
into  little  depressions,  while  at  still  other  points  the 
clear  gelatin  will  be  marked  by  more  or  less  saucer- 
shaped  pits  containing  opaque  fluid. 

Place  the  plate  containing  these  points  upon  the 
stage  of  a  microscope  and  examine  them  with  a  low- 
power  objective,  and  again  diiferences  will  be  observed. 
Some  of  these  minute  points  will  be  finely  granular, 
others  coarsely  so  ;  some  will  present  a  radiated  appear- 
ance, while  a  neighbor  may  be  concentrically  arranged  ; 

160 


THE  STUDY  OF  COLONIES.  161 

here  nothing  particularly  characteristic  will  present, 
there  the  point  may  resolve  itself  into  a  mass  having 
someAvhat  the  appearance  of  a  little  pellicle  of  raw  cot- 
ton. All  these  differences,  and  many  more,  aid  us  in 
saying  that  these  objects  must  be  different  in  their 
nature.  With  a  pointed  platinum  needle  take  up  a 
bit  of  one  of  these  small  islands,  prepare  it  for  micro- 
scopic examination  (see  chapter  on  Stained  Cover-slip 
Preparations),  and  examine  it  under  the  high-power  oil- 
immersion  objective,  with  access  of  the  greatest  amount 
of  light  afforded  by  the  illuminator  of  the  microscope. 
The  preparation  will  be  seen  to  be  made  up  entirely  of 
bodies  of  the  same  shape ;  they  will  all  be  spheres,  or 
ovals,  or  rods,  but  not  a  mixture  of  these  forms,  if  proper 
care  in  the  manipulation  has  been  taken.  Examine  in 
the  same  way  a  neighboring  spot  which  possesses  dif- 
ferent naked-eye  appearances,  and  it  will  often  be  found 
to  consist  of  bodies  of  an  entirely  different  appearance 
from  those  seen  in  the  first  preparation. 

These  spots  or  islands  on  the  surface  of  the  plates  are 
colonies  of  bacteria,  differing  severally,  not  only  in  their 
gross  appearances,  the  one  from  the  other,  but,  as  our 
cover-slip  preparations  show,  in  the  morphological  char- 
acteristics of  the  individual  organisms  composing  them. 

If  from  one  of  these  colonies  a  second  set  of  plates  be 
prepared,  the  peculiarities  which  were  first  observed  in 
it  will  be  reproduced  in  all  of  the  new  colonies  which 
develop ;  each  will  be  found  to  consist  of  the  same 
organisms  as  the  colony  from  which  the  plates  were 
made.  In  other  words,  these  peculiarities  are  constant 
under  uniform  conditions. 

The  appearance  of  the  colonies  developing  from 
all  organisms  is  regulated  by  their  location  in  the 
U 


162  BACTERIOLOGY. 

medium  in  which  they  are  growing.  When  deep 
down  in  the  medium  they  are  usually  round,  oval,  or 
lozenge-shaped ;  whereas  when  on  the  surface  of  the 
gelatin  or  agar  they  may  take  quite  a  different  form. 
This  is  purely  a  mechanical  effect  due  to  the  pressure 
of,  or  resistance  offered  by,  the  medium  surrounding 
them,  and  is  always  to  be  borne  in  mind,  otherwise 
errors  are  apt  to  arise. 

PURE  CULTURES. — If  from  one  .of  these  small  col- 
onies a  bit  be  taken  upon  the  point  of  a  sterilized  plati- 
num needle  and  introduced  into  a  tube  of  sterilized 
gelatin  or  agar-agar,  the  growth  that  results  will  be 
what  is  known  as  a  "  pure  culture,"  the  condition  to 
which  all  organisms  must  be  brought  before  a  system- 
atic study  of  their  many  peculiarities  is  begun.  Some- 
times several  series  of  plates  are  necessary  before  the 
organisms  can  be  obtained  pure,  but  by  patiently  fol- 
lowing this  plan  the  results  will  ultimately  be  satis- 
factory. 

TEST-TUBE  CULTURES  ;  STAB-CULTURES  ;  SMEAR- 
CULTURES. — After  separating  the  organisms  the  one 
from  the  other  by  the  plate  method  just  described,  they 
must  be  isolated  from  the  plates  as  pure  stab-  or  smear- 
cultures. 

This  is  done  in  the  following  way  :  decide  upon  the 
colony  from  which  the  pure  culture  is  to  be  made. 
Select  preferably  a  small  colony  and  one  as  widely  sep- 
arated from  other  colonies  as  possible.  Sterilize  in  a 
gas-flame  a  straight  platinum-wire  needle.  The  glass 
handle  of  the  needle  should  be  drawn  through  the  flame 
as  well  as  the  needle  itself,  otherwise  contamination  from 
this  source  may  occur.  When  it  is  cool,  which  is  in  five 
or  ten  seconds,  take  up  carefully  a  portion  of  the  colony. 


TEST-TUBE,  STAB-  AND  SMEAR-CULTURES.   163 

Guard  against  touching  anything  bid  the  colony.  If 
during  manipulation  the  needle  touches  anything  else 
whatever  than  the  colony  from  which  the  culture  is  to 
be  made,  it  must  be  sterilized  again.  This  holds  not 
only  for  the  time  before  touching  the  colony,  but  also 
during  its  passage  into  the  test-tube  from  the  colony ; 
otherwise  there  is  no  guarantee  that  the  growth  result- 
ing from  the  inoculation  of  this  bit  of  colony  into  a 
fresh  sterile  medium  will  be  pure. 

In  the  meantime  have  in  the  other  hand  a  test-tube 
of  sterile  medium  :  gelatin,  agar-agar,  or  potato.  This 
tube  is  held  across  the  palm  of  the  hand  in  an  almost 
horizontal  position  with  its  mouth  pointing  out  between 
the  thumb  and  index-finger  and  its  contents  toward  the 
body  of  the  worker.  With  the  disengaged  fingers  of  the 
other  hand  holding  the  needle  the  cotton  plug  is  removed 
from  the  tube  by  a  twisting  motion  and  placed  between 
the  index  and  second  fingers  of  the  hand  holding  the 
tube,  in  such  a  way  that  the  portion  of  the  plug  which 
fits  into  the  mouth  of  the  test-tube  looks  toward  the 
dorsal  surface  of  the  hand  and  does  not  touch  any  por- 
tion of  the  hand ;  this  is  accomplished  by  placing  only 
the  overhanging  portion  of  the  plug  between  the  fingers. 
The  needle  containing  the  bit  of  colony  is  now  to  be 
thrust  into  the  medium  in  the  tube  if  a  stab-culture  is 
desired,  or  rubbed  gently  over  its  surface  if  a  smear-  or 
stroke-culture  is  to  be  made.  The  needle  is  then  with- 
drawn, the  cotton  plug  replaced,  and  the  needle  sterilized 
before  it  is  laid  clown.  Neither  the  needle  nor  its 
handle  should  touch  the  inner  sides  of  the  test-tube  if  it- 
can  be  avoided.  The  tube  is  then  labelled  and  set  aside 
;  for  observation.  The  growth  which  appears  in  the  tube 
after  twenty-four  to  thirty-six  hours  should  be  a  pure 


164  BA  CTERWLOG  Y. 

culture   of   the   organisms    of   which    the    colony   was 
composed. 

Cultures  of  this  form  are  not  only  useful  as  a  means 
of  preserving  the  different  organisms  with  which  we 
may  be  working,  but  serve  also  to  bring  out  certain 

FIG.  33. 


Series  of  stab-cultures  in  gelatin,  showing  modes  of  growth  of  different 
species  of  bacteria. 

characteristics  of  different  organisms  when  grown  in 
this  way. 

If  gelatin  be  employed  and  the  organism  which  has 


TEST-TUBE,  STAB-  AND  SMEAR-CULTURES.    165 

been  introduced  into  it  possesses  the  power  of  bringing 
about  liquefaction — /.  <?.,  of  digesting  it — it  will  soon  be 
discovered  that  the  mode  of  liquefaction  differs  with 
different  organisms  and  is  practically  constant  for  the 
same  organism.  Some  bacteria  cause  a  liquefaction 
which  spreads  across  the  whole  upper  surface  of  the 
gelatin  and  continues  gradually  downward ;  with  others 
it  occurs  in  a  funnel-shape,  the  broad  end  of  the  funnel 
being  uppermost  and  the  point  downward,  correspond- 
ing to  the  track  of  the  needle;  at  times  a  stocking-  or 
sac-like  liquefaction  may  be  noticed.  (See  Fig.  33.) 

NOTE. — Obtain  a  number  of  organisms  from  different 
sources  in  pure  cultures  by  the  method  given.  Plant 
them  as  pure  cultures,  all  at  the  same  time,  in  gelatin — 
preferably  gelatin  of  the  same  making — retain  them 
under  the  same  conditions  of  temperature,  and  sketch 
the  finer  differences  in  the  way  in  which  liquefaction 
occurs. 

Select  from  your  collection  a  non-spore-bearing,  ac- 
tively liquefying  species.  Cultivate  it  as  a  pure  culture 
in  nutrient  bouillon  for  three  days.  Then  heat  this 
bouillon  culture  to  68°  C.  on  a  water-bath  for  ten 
minutes.  In  the  meantime  prepare  several  tubes  con- 
taining each  about  10  c.c.  of: 

Gelatin 7       grammes. 

Phenol 0.25  gramme. 

Water 100       c.c. 

Let  the  carbolized  gelatin  in  one  tube  remain  solid,  and 
bring  that  in  another  to  a  liquid  state  by  gentle  heat.  On 
the  surface  of  the  gelatin  in  the  first  tube  place  0.5  c.c.  of 
the  heated  (and  cooled)  culture,  and  mark  on  the  side  of 
the  tube  the  point  of  contact  between  the  fluid  culture  and 


166  BACTERIOLOGY. 

the  solid  gelatin.  To  the  tube  of  liquefied  gelatin  add 
likewise  0.5  c.c.  of  the  heated  culture,  mix  it  thoroughly 
with  the  gelatin,  and  place  the  tube  containing  the 
mixture  in  cold  water  until  the  mass  becomes  solid. 
Set  both  tubes  aside  at  a  temperature  not  above  20°  C. 
Note  what  occurs  at  the  end  of  an  hour,  by  next  day, 
and  after  three  days.  Alter  the  experiment  by  filtering 
the  three-day-old  bouillon  culture  through  a  porcelain 
or  a  Berkefeld  filter,  instead  of  heating  it  as  directed 
above.  Are  the  results  modified  ?  How  do  you  inter- 
pret these  results  ? 


CHAPTER  X. 

Methods  of  staining — Impression  cover-slip  preparations — Solutions 
employed — Preparation  and  staining  of  cover-slips — Staining 
solutions — Staining  in  general — Special  staining  methods. 

A  COMPLETE  list  of  solutions  and  methods  that  are 
recommended  for  the  staining  of  bacteria  is  not  essen- 
tial to  the  work  of  the  beginner,  so  that  only  those 
which  are  of  the  most  common  application  will  be  given 
in  this  book.  In  general,  it  suffices  to  say  that  bac- 
teria stain  best  with  watery  solutions  of  the  basic  ani- 
line dyes,  and  of  these,  fuchsin,  gentian- violet,  and 
methylene-blue  are  those  most  frequently  employed. 

In  practical  work  bacteria  are  either  dried  upon  cover- 
slips  and  then  stained,  or  stained  in  sections  of  tissues  in 
which  they  have  been  deposited  during  the  course  of 
disease.  In  both  processes  the  essential  point  to  be  borne 
in  mind  is  that  the  bacteria,  because  of  their  microscopic 
dimensions,  require  to  be  more  conspicuously  stained 
than  the  surrounding  materials  upon  the  cover-slips  or 
in  the  sections,  otherwise  their  recognition  is  a  matter  of 
the  greatest  difficulty,  if  not  of  impossibility.  For  this 
reason,  especially  in  section-staining,  it  frequently  be- 
comes necessary  to  decolorize  the  tissues  after  removing 
them  from  the  staining-solutions,  in  order  to  render  the 
bacteria  more  prominent,  and  for  this  purpose  special 
methods,  which  provide  for  decolorization  of  the  tissues 
without  robbing  the  bacteria  of  their  color,  are  employed. 
The  ordinary  method  of  cover-slip  examination  of  bac- 

167 


168  BACTERIOLOGY. 

teria,  constantly  in  use  in  these  studies,  is  performed  in 
the  following  way  : 

COVER-SLIP  PREPARATIONS. — In  order  that  the  dis- 
tribution of  the  organisms  upon  the  cover-slips  may 
be  uniform  and  in  as  thin  a  layer  as  possible  it  is  es- 
sential that  the  slips  should  be  clean  and  free  from 
grease.  For  cleansing  the  slips  several  methods  may 
be  employed. 

The  simplest  plan  with  new  cover-slips  is  to  immerse 
them  for  a  few  hours  in  strong  nitric  acid,  after  which 
they  are  rinsed  in  water,  then  in  alcohol,  then  ether, 
and,  finally,  they  may  be  kept  in  alcohol  to  which  a 
little  ammonia  has  been  added.  AYhen  about  to  be 
used  they  should  be  wiped  dry  with  a  clean  cotton  or 
silk  handkerchief. 

If  the  slips  have  been  previously  used,  boiling  in 
strong  soap  solution,  followed  by  rinsing  in  clean  warm 
water,  and  then  treating  as  above,  renders  them  clean 
enough  for  ordinary  purposes. 

A  method  commonly  employed  is  to  remove  all  coarse 
adherent  matter  from  slips  and  slides  by  allowing  them 
to  remain  for  a  time  in  strong  nitric  or  sulphuric  acid. 
They  are  removed  from  the  acid  after  several  days, 
rinsed  in  water,  and  treated  as  above.  Knauer  suggests 
the  boiling  of  soiled  cover-slips  and  slides  for  from 
twenty  to  thirty  minutes  in  a  10  per  cent,  watery  solu- 
tion of  lysol,  after  which  they  are  to  be  rinsed  carefully 
in  water  until  all  trace  of  the  lysol  has  disappeared. 
They  are  then  to  be  wiped  dry  with  a  clean  handker- 
chief. 

Loffler's  method,  which  provides  for  the  complete 
removal  of  all  grease,  is  to  warm  the  cover-slips  in  con- 
centrated sulphuric  acid  for  a  time  and  then  rinse  them 


COVER-SLIP  PREPARATIONS.  169 

in  water,  after  which  they  are  kept  in  a  mixture  of 
equal  parts  of  alcohol  and  ammonia.  They  are  to  be 
dried  on  a  cloth  from  which  all  fat  has  been  extracted. 

Steps  in  making  the  preparations.  Place  upon  the 
centre  of  one  of  the  clean  dry  cover-slips  a  very 
small  drop  of  water  or  physiological  salt-solution. 
With  a  platinum  needle,  which  has  been  sterilized  in 
a  gas-flame  just  before  using  and  allowed  to  cool,  take 
up  a  very  small  portion  of  the  colony  to  be  examined 
and  mix  it  carefully  with  the  drop  on  the  slip  until 
there  exists  a  very  thin  homogeneous  film  over  the 
larger  part  of  the  surface.  This  is  to  be  dried  upon 
the  slip  by  either  allowing  it  to  remain  upon  the  table 
in  the  horizontal  position  under  a  cover,  to  protect  it 
from  dust,  or  by  holding  it  between  the  fingers  (not  with 
forceps),  at  some  distance  above  a  gas-flame,  until  it 
is  quite  dry.  If  held  with  the  forceps  over  the  flame 
at  this  stage,  too  much  heat  may  be  unconsciously  ap- 
plied, and  the  morphology  of  the  organisms  in  the  prep- 
aration distorted.  When  held  between  the  fingers 
with  the  thin  layer  of  bacteria  away  from  the  flame  no 
such  accident  is  likely  to  occur.  When  the  whole 
pellicle  is  completely  dried  the  slip  is  to  be  taken  up 
with  forceps,  and,  holding  the  side  upon  which  the  bac- 
teria are  deposited  away  from  the  direct  action  of  the 
flame,  it  is  to  be  passed  through  the  flame  three  times, 
a  little  more  than  one  second  being  allowed  for  each 
transit.  Unless  the  preliminary  drying  at  the  low  tem- 
perature has  been  complete,  the  preparation  will  be 
rendered  worthless  by  the  subsequent  "  fixing "  at  the 
higher  temperature,  for  the  reason  that  the  protoplasm 
of  bacteria  when  moist  coagulates  at  these  tempera- 
tures, and  in  doing  so  the  normal  outline  of  the  cells  is 


170  BACTERIOLOGY. 

altered.  If  carefully  dried  before  fixing,  this  does  not 
occur  and  the  morphology  of  the  organism  remains  un- 
changed. 

A  better  plan  for  the  process  of  fixing  is  to  employ 
a  copper  plate  about  35  cm.  long  by  10  cm.  wide  by 
0.3  cm.  thick.  This  plate  is  laid  upon  an  iron  tripod 
and  a  small  gas-flame  is  placed  beneath  one  of  its 
extremities.  By  this  arrangement  one  can  get  a  gradu- 
ated temperature,  beginning  at  the  part  of  the  plate 
above  the  gas-flame  where  it  is  hottest,  and  becom- 
ing gradually  cooler  toward  the  other  end  of  the  plate, 
which  may  be  of  a  very  low  temperature.  By  dropping 
water  upon  the  plate,  beginning  at  the  hottest  point  and 
proceeding  toward  the  cooler  end,  it  is  easy  to  determine 
the  point  at  which  the  water  just  boils ;  it  is  at  a  little 
below  this  point  that  the  cover-slips  are  to  be  placed, 
bacteria  side  up,  and  allowed  to  remain  about  ten  min- 
utes, when  the  fixing  will  be  complete.  The  same  may 
be  accomplished  in  a  small  copper  drying-oven,  which 
is  regulated  to  remain  at  a  temperature  of  from  95°  to 
98°  C.  In  very  particular  comparative  studies  this  plan 
is  to  be  preferred  to  the  process  of  passing  the  cover- 
slips  through  a  flame,  as  the  organisms  are  always  sub- 
jected to  the  same  degree  of  heat,  and  the  distortions 
which  sometimes  occur  from  too  great  and  irregular  ap- 
plication of  high  temperatures  may  be  eliminated.  The 
fixing  consists  in  drying  or  coagulating  the  gelatinous  en- 
velope surrounding  the  organisms,  by  which  means  they 
are  caused  to  adhere  to  the  surface  of  the  cover-slip.  It 
is  sometimes  desirable  to  fix  the  preparations  without  the 
use  of  heat,  as  in  the  case  of  pus  or  other  exudates.  In 
this  event,  after  drying  the  thinly  spread  material  care- 
fully in  the  air,  the  cover-slip  on  which  it  is  placed  is 


COVER-SLIP  PREPARATIONS.  171 

immersed  in  a  mixture  of  equal  parts  of  absolute  alco- 
hol and  ether  for  about  15  minutes.  At  the  end  of 
this  time  it  may  be  removed  and  stained.  The  advan- 
tage of  this  method  is  that  there  is  less  distortion  and, 
as  a  rule,  less  precipitation  (or,  perhaps  better,  no  char- 
ring) of  extraneous  matter.  When  fixed,  staining  is 
usually  a  simple  matter.  The  majority  of  bacteria  with 
which  the  beginner  will  have  to  deal  stain  readily  with 
watery  solutions  of  any  of  the  basic  aniline  dyes,  such, 
for  instance,  as  fuchsin,  methylene-blue,  or  gentian- 
violet. 

To  stain  the  fixed  cover-slip  preparation,  it  is  taken 
by  one  of  its  edges  between  forceps,  and  a  few  drops 
of  a  watery  solution  of  either  of  the  dyes  named  are 
placed  upon  the  film  and  allowed  to  remain  twenty 
to  thirty  seconds.  The  slip  is  then  carefully  rinsed 
in  water,  and  without  drying  is  placed  bacteria  down 
upon  a  slide ;  the  excess  of  water  is  taken  up  by 
covering  it  with  blotting-paper  and  gently  pressing 
upon  it,  after  which  the  preparation  is  ready  for  ex- 
amination. 

Another  plan  sometimes  used  is  to  bring  the  slip 
upon  the  slide,  bacteria  down,  without  rinsing  oif  the 
staining-fluid  ;  the  excess  of  fluid  is  removed  with 
blotting-paper  and  the  preparation  is  ready  for  exam- 
ination with  the  microscope.  This  method  is  satis- 
factory and  time-saving,  but  must  always  be  prac- 
tised with  care.  The  staining-fluid  should  always  be 
filtered  before  using,  to  rid  it  of  insoluble  particles 
which  might  be  taken  for  bacteria.  If  upon  exami- 
nation the  preparation  proves  of  particular  interest, 
so  that  it  is  desirable  to  preserve  it,  then  it  may  be 
mounted  permanently.  The  drop  of  immersion  oil 


172  BACTERIOLOGY. 

is  to  be  removed  from  the  surface  of  the  slip  with 
blotting-paper,  and  the  slip  loosened,  or  rather  floated, 
from  the  slide  by  allowing  water  to  flow  around  its 
edges.  It  is  then  taken  up  with  forceps,  carefully  de- 
prived of  the  water  adhering  to  it  by  means  of  blotting- 
paper,  and  allowed  to  dry.  When  dry  it  is  mounted  in 
xylol-Canada-balsam  by  placing  a  small  drop  of  the 
balsam  upon  the  surface  of  the  film,  and  then  inverting 
the  slip  upon  a  clean  glass  slide.  It  is  sometimes  de- 
sirable to  have  the  balsam  harden  quickly,  and  a  method 
that  is  commonly  employed  to  induce  this  is  as  follows : 
the  slide,  held  by  one  of  its  ends  between  the  fingers,  is 
warmed  over  a  gas-flame  until  quite  hot ;  a  drop  of 
balsam  is  then  placed  on  the  centre  of  it,  and  it  is  again 
warmed ;  the  cover-slip  is  then  placed  in  position,  and 
when  the  balsam  is  evenly  distributed  the  temperature 
is  rapidly  reduced  by  rubbing  the  bottom  of  the  slide 
with  a  towel  wet  with  cold  water.  Usually  the  prepara- 
tion is  firmly  fixed  after  this  treatment ;  a  little  practice 
is  necessary  j  however,  in  order  not  to  overheat  and 
crack  the  slide.  The  method  is  applicable  only  to 
cover-slip  preparations,  and  cannot  be  safely  used  with 
tissues. 

IMPRESSION  COVER-SLIP  PREPARATIONS. — Impres- 
sion preparations  differ  from  ordinary  cover-slip  prep- 
arations in  only  one  respect :  they  present  an  impression 
of  the  organisms  as  they  were  arranged  in  the  colony 
from  which  the  preparation  is  made.  They  are  made 
by  gently  covering  the  colony  with  a  thin,  clean  cover- 
slip,  lightly  pressing  upon  it,  and,  without  moving  the 
slip  laterally,  lifting  it  by  one  of  its  edges.  The  organ- 
isms adhere  to  the  slip  in  the  same  relation  to  one 
another  that  they  had  in  the  colony.  The  subsequent 


ORDINARY  STAINIXG-SOLUTIONS.  173 

steps  of  drying,  fixing,  staining,  and  mounting  are  the 
same  as  those  just  given  for  ordinary  cover-slip  prep- 
arations. 

By  this  method  constancies  in  the  arrangement  and 
grouping  of  the  individuals  in  a  colony  can  often  be 
made  out.  Some  will  always  appear  irregularly  massed, 
others  show  growth  in  parallel  bundles,  while  others, 
again,  will  be  seen  as  long,  twisted  threads. 

NOTE. — From  a  colony  of  bacillus  subtilis  make  a 
cover-slip  preparation  in  the  ordinary  way  ;  now  make 
an  impression  cover-slip  preparation  of  another  colony 
of  the  same  organism.  Compare  the  results. 

ORDINARY  STAINING-SOLUTIONS.  —  The  solutions 
commonly  employed  in  staining  cover-slip  preparations 
are,  as  has  been  stated,  watery  solutions  of  the  basic 
aniline  dyes — fuchsin,  gentian-violet,  and  methylene- 
blue.  These  solutions  may  be  made  either  by  directly 
dissolving  the  dyes  in  substance  in  water  until  the 
proper  degree  of  concentration  has  been  reached,  or  by 
using  concentrated  watery  or  alcoholic  solutions  of  the 
dyes  which  may  be  kept  on  hand  as  stock.  The  latter 
method  is  the  one  commonly  practised. 

The  solutions  of  the  colors  which  are  in  constant  use 
in  staining  are  prepared  as  follows : 

Prepare  as  stock,  saturated  alcoholic  or  watery  solu- 
tions of  fuchsin,  gentian- violet,  and  methylene-blue. 
These  solutions  are  best  made  by  pouring  into  clean 
bottles  enough  of  the  dyes  in  substance  to  fill  them  to 
about  one-fourth  of  their  capacity.  Each  bottle  should 
then  be  filled  with  alcohol  or  with  water,  tightly  corked, 
well  shaken,  and  allowed  to  stand  for  twenty-four  hours. 


174  BACTERIOLOGY. 

If  by  then  all  the  staining-material  has  been  dissolved, 
more  should  be  added,  the  bottle  being  again  shaken 
and  allowed  to  stand  for  another  twenty-four  hours ; 
this  must  be  repeated  until  a  permanent  sediment  of 
uudissolved  coloring-matter  is  seen  upon  the  bottom 
of  the  bottle.  The  bottles  are  then  to  be  labelled 
"  saturated  alcoholic  "  or  "  watery  "  solution  of  fuch- 
sin,  gentian-violet,  or  methylene-blue,  as  the  case  may 
be.  The  alcoholic  solutions  are  not  directly  employed  for 
staining-purposes. 

The  solutions  with  which  staining  is  accomplished 
are  made  from  the  stock  solutions  in  the  following 
way  : 

An  ordinary  test-tube  of  about  13  mm.  diameter  is 
three-fourths  filled  with  distilled  water  and  the  con- 
centrated alcoholic  or  watery  solution  of  the  dye 
added,  little  by  little,  until  one  can  just  see  through 
the  solution.  It  is  then  ready  for  use.  Care  must  be 
taken  that  the  color  does  not  become  too  dense.  The 
best  results  are  obtained  when  this  dilution  is  just 

FIG.  34. 


Rack  of  bottles  for  staining-solutions. 

transparent  as  viewed   through   a   layer   about    12  to 
14  mm,  thick.     A  solution  consisting  of  5  parts  of  the 


ORDINARY  STA1NING-SOLUTIONS.  175 

saturated  alcoholic  solution  and  95  parts  of  distilled 
water  proves,  as  a  general  thing,  to  be  satisfactory. 

The  above  represent  the  staining-solutions  in  every- 
!  day  use.  They  may  be  kept  in  bottles  supplied  with 
stoppers  and  pipettes  (Fig.  34),  and  when  used  are 
dropped  upon  the  preparation  to  be  stained. 

For  certain  bacteria  which  stain  only  imperfectly 
with  these  simple  solutions  it  is  necessary  to  employ 
some  agent  that  will  increase  the  penetrating  action  of 
\  the  dyes.  Experience  has  taught  us  that  this  can  be 
accomplished  by  the  addition  to  the  solutions  of  small 
quantities  of  alkaline  substances,  or  by  dissolving  the 
staining-materials  in  strong  watery  solutions  of  either 
aniline  oil  or  carbolic  acid,  instead  of  water — in  other 
words,  by  employing  special  solvents  and  mordants 
with  the  stains. 

Of  the  solutions  thus  prepared  which  may  always  be 
employed  upon  bacteria  that  show  a  tendency  to  stain 
imperfectly,  there  are  three  in  common  use — Loffler's 
alkaline  methylene-blue  solution ;  the  Koch-Ehrlich 
aniline-water  solution  of  either  fuchsin,  gentian-violet, 
or  methylene-blue  ;  and  Ziehl's  solution  of  fuchsin  in 
carbolic  acid.  These  solutions  are  as  follows  : 

Loffler's  alkaline  methylene-blue  solution  : 

Concentrated  alcoholic  solution  of  methylene-blue  .    .      30  c.c. 
Caustic  potash  in- 1 : 10,000  solution 100  c.c. 

Koch-Ehrlich  aniline-water  solution.  To  about  100 
c.c.  of  distilled  water  aniline  oil  is  added  drop  by  drop 
until  the  solution  has  an  opaque  appearance,  the  vessel 
loontaining  the  solution  being  thoroughly  shaken  after 
i the  addition  of  each  drop.  It  is  then  filtered  through 
moistened  filter-paper  until  the  filtrate  is  perfectly 


176  BA  CTERIO  LOGY. 

clear.     To  100  c.c.  of  the  clear  filtrate  add  10  c.c.  of 
absolute  alcohol  and   11  c.c.  of  the  concentrated  alco- 
holic solution  of  either  fuchsin,  methylene-blue,  or  gen- 
tian-violet, preferably  fuchsin  or  gentian-violet. 
ZiehVs  carbol-fuchsin  solution  : 

Distilled  water 100  c.c. 

Carbolic  acid  (crystallized) 5  grammes. 

Alcohol 10  c.c. 

Fuchsin  in  substance 1  gramme. 

Or  it  may  be  prepared  by  adding  to  a  5  per  cent, 
watery  solution  of  carbolic  acid  the  saturated  alcoholic 
solution  of  fuchsin  until  a  metallic  lustre  appears  on 
the  surface  of  the  fluid. 

The  Koch-Ehrlich  solution  decomposes  after  a  time, 
so  that  it  is  better  to  prepare  it  fresh  in  small  quantities 
when  needed  than  to  employ  old  solutions.  Solution 
older  than  fourteen  days  should  not  be  used. 

The  three  solutions  just  given  maybe  used  for  cover- 
glass  preparations  in  the  ordinary  way. 

In  some  manipulations  it  becomes  necessary  to  stain 
the  bacteria  very  intensely,  so  that  they  may  retain 
their  color  when  exposed  to  the  action  of  decolorizing 
agents.  These  methods  are  usually  employed  when  it 
is  desirable  to  deprive  surrounding  objects  or  tissues  of 
their  color,  in  order  that  the  stained  bacteria  may  stand 
out  in  greater  contrast.  It  is  in  these  cases  that  the 
staining-solution  with  which  the  bacteria  are  being 
treated  is  to  be  warmed,  and  in  some  cases  boiled,  so  as 
further  to  increase  its  penetrating  action.  When  so 
treated,  certain  of  the  bacteria  will  retain  their  color, 
even  when  exposed  to  very  strong  decolorizers.  The 
tubercle  bacillus  is  distinguished  from  the  great  ma- 


STAINING  JN  GENERAL.  Ill 

jority  of  other  bacteria  by  the  tenacity  with  which  it 
retains  the  color  when  treated  in  this  way ;  it  is  an 
organism  difficult  to  stain,  but  when  once  stained  is 
equally  difficult  to  rob  of  its  color. 

STAINING    IN    GENERAL. 

The  physics  of  staining  and  decolorization  is  hardly 
a  subject  to  be  discussed  at  length  in  a  book  of  this 
character ;  but,  as  Kuhne  has  pointed  out,  it  may  be 
briefly  said  that  solutions  which  favor  the  production  of 
diifusion-currents  facilitate  intensity  of  staining,  and  by 
a  similar  process  increase  the  energy  of  decolorizing- 
agents.  For  example,  tissues  which  are  transferred 
from  water  into  watery  solutions  of  the  coloring-mat- 
ters are  less  intensely  stained  and  more  easily  decolor- 
ized than  when  transferred  from  alcohol  into  watery 
staining-fluids ;  for  the  same  reason  tissues  stained  in 
watery  solutions  of  the  dyes  do  not  become  decolorized 
so  readily  when  placed  in  water  as  when  placed  in 
alcohol. 

The  diffusion  of  staining-solutions  in  the  protoplasm 
of  dried  bacteria,  as  found  upon  cover-slip  preparations, 
is  much  greater  and  more  rapid  than  when  the  same 
bacteria  are  located  in  the  interstices  of  tissues.  These 
differences  are  not  in  the  bacteria  themselves,  but  in  the 
obstruction  to  diffusion  offered  by  the  tissues  in  which 
they  are  located.  The  result  of  absence  of  diffusion 
may  easily  be  illustrated  : 

Prepare  a  cover-slip  preparation,  dry  it  carefully,  fix 

it,  and,  without  allowing  water  to  get  on  it  from  any 

source,   attempt   to   stain   it   with    a   solution    of    the 

;  dyes  in  absolute  alcohol,  washing  it  subsequently  with 

12 


178  BACTERIOLOGY, 

absolute  alcohol ;  the  result  is  negative.  The  abso- 
lute alcohol  does  not  possess  the  property  of  diffusing 
into  the  dried  tissues,  and  hence,  as  has  been  stated, 
alcoholic  solutions  of  the  stain  ing-dyes  cannot  be  satis- 
factorily employed.  The  stain  ing-dyes  should  always 
be  watery.1 

DECOLORIZING-SOLUTIONS. — As  regards  the  employ- 
ment of  deeolorizing-agents,  it  must  always  be  borne  in 
mind  that  objects  which  are  easily  stained  are  also  easily 
decolorized,  and  those  that  can  be  made  to  take  up  the 
staining-material  only  with  difficulty  are  also  very  diffi- 
cult to  rob  of  their  color.  The  most  common  decolor- 
izer  in  use  is  probably  alcohol — not  absolute  alcohol, 
but  alcohol  containing  more  or  less  of  water.  Water 
alone  has  this  property,  but  in  a  much  less  degree  than 
dilute  alcohol.  On  the  other  hand,  a  much  more  ener- 
getic decolorization  than  that  possessed  by  either  alone 
can  be  obtained  by  alternate  exposures  to  alcohol  and 
water.  More  energetic  in  their  decolorizing  action  than 
either  water  or  alcohol  are  solutions  of  the  acids.  They 
appear,  particularly  when  they  are  alcoholic  solutions, 
to  diifuse  rapidly  into  tissues  and  bacteria  and  very 
quickly  extract  the  staining-materials  which  have  been 
deposited  there.  For  this  reason  these  solutions  should 
be  employed  with  much  care. 

Very  dilute  acetic  acid  robs  tissues  and  bacteria  of 
their  stain  with  remarkable  activity ;  still  more  ener- 
getic are  solutions  of  the  mineral  acids,  and  particularly, 

1  In  the  beginning  of  this  chapter  it  was  stated  that  the  saturated 
alcoholic  solutions  of  the  dyes  do  not  serve  as  stains  for  bacteria.  It 
must  be  remembered  that  this  holds  only  when  absolute  alcohol  and 
perfectly  dry  coloring-matters  have  been  used.  If  but  a  small  propor- 
tion of  water  is  present,  the  bacteria  may  be  stained  with  these  solu- 
tions, though  the  results  are,  as  a  rule,  unsatisfactory. 


STAINING   THE  TUBERCLE  BACILLUS.         179 

•as  lias  been  said,  when  this  action  is  accompanied  by 
the  decolorizing-properties  of  alcohol. 

The  acid  solutions  commonly  employed  are  : 

Acetic  acid  in  from  0.1  to  5  per  cent,  watery  solution. 

Nitric  acid  in  from  20  to  30  per  cent,  watery  solu- 
tion. 

Sulphuric  acid  in  from  5  to  10  per  cent,  solution  in 
water. 

Hydrochloric  acid  in  from  1  to  3  per  cent,  solution  in 
alcohol. 

NOTE. — For  details  as  to  the  technique  of  hardening 
and  cutting  sections  and  staining  bacteria  in  tissues,  the 
student  is  referred  to  Mallory  and  Wright's  Pathological 
Technique. 

METHOD  OF  STAINING  THE  TUBEECLE  BACILLUS. 
— Select  from  the  sputum  of  a  tuberculous  subject  one 
of  the  small,  white,  cheesy  masses  which  it  is  seen 
to  contain.  Spread  this  upon  a  cover-slip,  dry  and 
fix  it  in  the  usual  way.  The  slip  is  now  to  be  taken 
by  its  edge  with  forceps  and  the  film  covered  with  a 
few  drops  of  either  the  solution  of  Koch-Ehrlich  or 
that  of  Ziehl.  It  is  then  held  over  a  gas-flame,  at  first 
some  distance  away,  gradually  being  brought  nearer 
until  the  fluid  begins  to  boil.  After  it  has  bubbled 
once  or  twice  it  is  removed  from  the  flame,  the  excess 
of  stain  washed  away  in  a  stream  of  water,  then  im- 
mersed in  a  30  per  cent,  solution  of  nitric  acid  in 
water,  and  allowed  to  remain  until  all  color  has  dis- 
appeared. This  takes  longer  in  some  cases  than  in 
others.  One  can  always  determine  if  decolorization  is 
complete  by  washing  off  the  acid  in  a  stream  of  water. 
If  the  preparation  is  still  distinctly  colored,  it  should 
be  again  immersed  in  the  acid ;  if  of  only  a  very  faint 


180  BACTERIOLOGY. 

color,  it  may  be  dipped  in  alcohol,  again  washed  in 
water,  and  stained  with  some  contrast-color.  If,  for 
example,  the  tubercle  bacilli  have  been  stained  with 
fuchsin,  methylene-blue  forms  a  good  contrast-stain. 
In  making  the  contrast-stain  the  steps  in  the  process 
are  exactly  those  followed  in  the  ordinary  staining 
of  cover-slip  preparations  in  general :  the  slip  contain- 
ing the  stained  tubercle  bacilli  is  carefully  rinsed  in 
water,  and  a  few  drops  of  the  methylene-blue  solution 
placed  upon  it  and  allowed  to  remain  for  thirty  or 
forty  seconds,  when  it  is  again  rinsed  in  water  and 
examined  microscopically.  For  this  purpose  of  observ- 
ing the  difference  in  behavior  of  the  tubercle  bacilli 
and  the  other  organisms  present  in  the  preparation 
toward  this  method  of  staining,  it  is  well  to  exam- 
ine the  preparation  microscopically  before  the  con- 
trast-stain is  made;  then  give  it  the  contrast-color, 
and  again  examine.  It  will  be  seen  that  before  the 
contrast-color  has  been  given  to  the  preparation  the 
tubercle  bacilli  are  the  only  stained  objects  to  be 
made  out,  and  the  preparation  appears  devoid  of 
other  organisms ;  but  upon  examining  it  after  it  has 
received  the  contrast-color  a  great  many  other  or- 
ganisms will  appear;  these  take  on  the  second  color 
employed,  while  the  tubercle  bacilli  retain  their  orig- 
inal color.  Before  decolorization  all  organisms  in  the 
preparation  were  of  the  same  color,  but  during  the  appli- 
cation of  the  decolorizing  solution  all  except  the  tubercle 
bacilli  gave  up  their  color.  This  micro-chemical  charac- 
teristic, together  with  reactions  to  be  described,  serves 
to  differentiate  the  tubercle  bacillus  from  other  organ- 
isms with  which  it  might  be  confounded.  A  number 
of  different  methods  have  been  suggested  for  the  stain- 


STAINING   THE  TUBERCLE  BACILLUS.        181 

ing  of  tubercle  bacilli,  but  the  original  method  as  em- 
ployed by  Koch  is  so  satisfactory  in  its  results  that  it  is 
not  advisable  to  substitute  others  for  it.  The  above 
differs  from  the  original  Koch-Ehrlich  method  for  the 
staining  of  tubercle  bacilli  in  sputum  only  in  the  occa- 
sional employment  of  ZiehPs  carbol-fuchsin  solution 
and  in  the  method  of  heating  the  preparation  with  the 
staining-fluid  upon  it. 

As  Nuttall  has  pointed  out,  however,  the  strong  acid 
decolorizer  used  in  this  method  can,  with  advantage, 
be  replaced  by  much  more  dilute  solutions,  as  a  number 
of  the  bacilli  are  entirely  decolorized  by  the  too  energetic 
action  of  the  strong  acids.  He  recommends  the  follow- 
ing method  of  decolorization :  after  staining  the  slip  or 
section  in  the  usual  way,  pass  it  through  three  alcohols ; 
it  is  then  to  be  washed  in  a  solution  composed  of 

Water 150  c.c. 

Alcohol 50  c.c. 

Concentrated  sulphuric  acid   ........      20  to  30  drops. 

From  this  it  is  removed  to  water  and  carefully  rinsed. 
The  remaining  steps  in  the  process  are  the  same  as  those 
given  in  the  other  methods. 

GABBETT'S  METHOD  for  the  staining  of  tubercle 
bacilli  recommends  itself  because  of  its  simplicity  and 
the  rapidity  with  which  it  can  be  performed.  By  many 
it  is  considered  the  best  method  for  routine  employ- 
ment. It  consists  in  staining  the  cover-slips,  prepared 
in  the  manner  given,  for  from  two  to  five  minutes  in 
a  cold  carbol-fuchsin  solution,  after  which  they  are  sub- 
jected to  the  action  of  Gabbett's  methylene-blue  sul- 
phuric acid  solution.  This  latter  consists  of 

Sulphuric  acid  (strength  25  per  cent.)    ....  100  c.c. 
Methylene-blue,  in  substance 1  to  2  grammes. 


182  BACTERIOLOGY. 

The  cover-slips  are  then  rinsed  in  water  and  are  ready 
for  examination.  The  tubercle  bacilli  will  be  stained 
red  by  the  fuchsin,  while  all  other  bacteria,  cell-nuclei, 
etc.,  will  be  tinted  blue. 

GRAM'S  METHOD. — Another  important  differential 
method  of  staining  which  is  very  commonly  employed 
is  that  recommended  by  Gram.  In  this  method  the 
objects  are  treated  with  an  aniline-water  solution  of 
gentian- violet  made  after  the  formula  of  Koch-Ehrlich. 
After  remaining  in  this  for  twenty  to  thirty  minutes 
they  are  immersed  in  a  solution  composed  of 

Iodine 1  gramme. 

Potassium  iodide 2  grammes. 

Distilled  water 300  c.c. 

In  this  they  remain  for  about  five  minutes ;  they  are 
then  transferred  to  alcohol  and  thoroughly  rinsed.  If 
still  of  a  violet  color,  they  are  again  treated  with  the 
iodine  solution,  followed  by  alcohol,  and  this  is  con- 
tinued until  no  trace  of  violet  is  visible  to  the  naked 
eye.  They  may  then  be  examined,  or  a  contrast-color 
of  carmine  or  Bismarck-brown  may  be  given  them. 

This  method  is  particularly  useful  in  demonstrating 
the  capsule  which  is  seen  to  surround  some  bacteria, 
especially  micrococcus  lanceolatus  of  pneumonia. 

GLACIAL  ACETIC  ACID  METHOD. — Another  method 
that  may  be  employed  for  demonstrating  the  presence 
of  the  capsule  surrounding  certain  organisms  is  to  pre- 
pare the  cover-slips  in  the  ordinary  way,  then  cover  the 
layer  of  bacteria  upon  them  with  glacial  acetic  acid, 
which  is  instantly  poured  off  (not  washed  oif  in  water), 
and  the  aniline-water  gentian- violet  solution  dropped 
upon  them ;  this  is  allowed  to  remain  three  or  four 


STAINING   OF  SPORES.  183 

minutes,  is  poured  off,  and  a  few  drops  more  are  added, 
and  lastly  the  slip  is  washed  in  a  solution  of  sodium 
chloride.  Usually  this  is  of  the  strength  of  physio- 
logical salt-solution,  viz.,  0.6  to  0.7  per  cent. ;  but  at 
times  it  must  be  stronger,  occasionally  as  concen- 
trated as  1.5  to  2  per  cent.  The  reason  for  this 
is  that  if  the  slips  be  washed  in  water,  or  in  salt- 
solution  that  is  too  weak,  the  mucin  capsule  that 
has  been  coagulated  by  the  acetic  acid  is  redissolved 
and  rendered  invisible.  This  does  not  occur  when  the 
salt-solution  is  of  the  proper  strength — a  point  that  can 
be  determined  only  after  a  few  trials  with  solutions  of 
different  strengths.  (Welch.)  A  very  clear,  sharply  cut 
picture  usually  follows  this  method  of  procedure. 

Ribbert  also  recommends  for  the  staining  of  capsu- 
lated  bacteria  the  momentary  immersion  of  the  cover- 
slips  in  a  saturated  solution  of  dahlia  in  a  mixture  of 
100  parts  of  water,  50  parts  of  alcohol,  and  12^  parts 
of  glacial  acetic  acid ;  after  which  the  excess  of  color 
is  removed  by  washing  in  wajter^.vv^' 

STAINING  OF  SPORES. — We*^*ce  learned  that  one  of 
the  points  by  which  spores  may  be  recognized  is  their 
refusal  to  take  up  staining-substances  when  applied  in 
the  ordinary  way.  They  may,  however,  be  stained  by 
special  methods ;  of  these,  one  that  has  given  fairly  satis- 
factory results  in  our  hands  is  as  follows :  the  cover- 
slip  is  to  be  prepared  from  the  material  containing  the 
spores  in  the  ordinary  way,  dried,  and  fixed.  It  is  then 
to  be  held  by  its  edge  with  forceps,  and  its  surface  cov- 
ered with  Loftier' s  alkaline  methylene-blue  solution.  It 
is  then  held  over  the  Bunsen  flame  until  the  fluid  boils  ; 
it  is  then  removed,  and  after  a  few  seconds  is  heated 
again.  This  is  continued  for  about  one  minute,  after 
which  it  is  washed  in  water  and  then  decolorized  in 


184  BACTERIOLOGY. 

Alcohol  (80  per  cent.)      98  c.c. 

Nitric  acid  9 


until  all  visible  blue  color  has  disappeared.     It  is  then 
rinsed  in  water  and  dipped  for  from  3  to  5  seconds  in 

Saturated  alcoholic  solution  of  eosin 10  c.c. 

Water      90  c.c. 

after  which  it  is  again  rinsed  in  water  and  finally 
mounted  for  examination.  If  the  decolorization  in 
the  acid  alcohol  be  not  carried  too  far,  the  preparation 
will  show  the  spores  stained  blue  and  the  bodies  of  the 
cells  to  have  taken  on  the  rose  color  characteristic  of  eosin. 
By  another  process  the  cover-slip  is  floated,  bacteria 
down,  upon  the  surface  of  freshly  prepared  Koch- 
Ehrlich  solution  of  fuchsin  contained  in  a  watch-crys- 
tal. This  is  then  held  by  its  edge  with  forceps  about 
2  cm.  above  a  very  small  flame  of  a  Bunsen  burner, 
care  being  taken  that  the  flame  touches  only  the  centre 
of  the  bottom  of  the  crystal.  After  a  few  seconds  the 
crystal  is  gradually  elevated  until  it  is  about  6  to  8  cm. 
above  the  flame ;  then  it  is  slowly  moved  down  to  the 
flame  again,  and  this  up-and-down  movement  is  con- 
tinued until  the  staining-fluid  begins  to  boil.  As  soon 
as  a  few  bubbles  have  been  given  off  it  is  held  aside 
for  a  minute  or  two,  when  the  heating  is  repeated. 
When  the  boiling  begins  the  crystal  is  again  held  aside 
for  a  minute  or  two.  The  crystal  is  heated  in  this  way 
five  or  six  times.  When  the  fluid  has  stood  for  about  five 
minutes  after  the  last  boiling  the  preparation  is  trans- 
ferred, without  washing  in  water,  to  a  second  watch- 
crystal  containing  the  following  decolorizing  solution  : 

Absolute  alcohol 100  c.c. 

Hydrochloric  acid 3  c.c. 


MOELLER'S  METHOD  FOR  STAINING  SPORES.    185 

In  this  solution  it  is  placed,  bacteria  up,  and  the 
vessel  is  tilted  from  side  to  side  for  about  one  minute. 
It  is  then  removed,  washed  in  water,  and  stained  with 
the  methylene-blue  solution.  The  spores  will  be  stained 
red  and  the  body  of  the  cells  blue. 

MOELLER'S  METHOD  FOR  STAINING  SPORES. — A 
method  that  has  recently  been  published  by  Moeller 
is  designed  to  favor  the  penetration  of  the  coloring- 
material  through  the  spore-membrane  by  macerating 
the  spores  in  a  solution  of  chromic  acid  before  staining 
them.  It  is  as  follows  : 

The  cover-slips  are  prepared  in  the  usual  way,  or  the 
fixing  may  be  accomplished  with  absolute  alcohol  in- 
stead of  high  temperatures.  The  preparation  is  held 
for  two  minutes  in  chloroform,  washed  in  water,  placed 
for  from  one-half  to  two  minutes  in  a  5  per  cent,  solu- 
tion of  chromic  acid,  again  washed  in  water,  and  stained 
with  carbol-fuchsin.  In  the  process  of  staining,  the  slip 
is  taken  by  the  corner  with  forceps,  and  carbol-fuchsin 
is  dropped  upon  the  side  containing  the  spores.  It  is 
then  held  over  a  flame  until  it  boils,  and  then  held 
some  distance  above  the  flame  for  one  minute.  The 
staining-fluid  is  then  poured  off  and  the  preparation  is 
completely  decolorized  in  5  per  cent,  sulphuric  acid, 
again  washed  in  water,  and  finally  stained  for  thirty 
seconds  in  the  watery  methylene-blue  solution.  The 
spores  will  be  red,  the  body  of  the  cells  blue. 

In  this  method  the  object  of  the  preliminary  ex- 
posure to  chloroform  is  to  dissolve  any  crystals  of  leci- 
thin, cholesterin,  or  fat  that  may  be  in  the  preparation, 
and  which  when  stained  might  cause  confusion. 

It  must  be  remembered  that  there  are  conspicuous 
differences  in  the  behavior  of  spores  of  different  bacteria 
to  staining-methods  and  of  the  spores  of  a  single  species 


186  BACTERIOLOGY. 

in  different  stages  of  development.  Some  stain  readily 
by  either  of  the  methods  especially  devised  for  this 
purpose,  while  others  ean  hardly  be  stained  at  all,  or 
only  with  the  greatest  difficulty,  by  any  of  the  known 
processes ;  some  stain  readily  when  fully  developed,  but 
with  difficulty  when  only  partly  developed  ;  others  have 
this  peculiarity  reversed. 

LOFFLER'S  METHOD  FOR  STAINING  FLAGELLA.— - 
For  the  demonstration  of  the  locomotive  apparatus  pos- 
sessed by  motile  bacteria  we  are  indebted  to  Loffler. 
By  a  special  method  of  staining,  in  which  the  use  of 
mordants  played  the  essential  part,  he  has  shown  that 
these  organisms  possess  very  delicate,  hair-like  appen- 
dages, by  the  lashing  movements  of  which  they  propel 
themselves  through  the  fluid  in  which  they  are  growing. 
The  method  as  given  by  Loffler  is  as  follows : 

It  is  essential  that  the  bacteria  be  evenly  and  not 
too  numerously  distributed  upon  the  cover-slip.  The 
slips  must  therefore  be  perfectly  clean.  (See  Loffler's 
method  of  cleaning  cover-slips.)  Five  or  six  of  the 
carefully  cleansed  cover-slips  are  to  be  placed  in  a  line 
on  a  table,  and  on  the  centre  of  each  slip  a  very  small 
drop  of  tap-water  is  placed.  From  the  culture  to  be 
examined  a  minute  portion  is  transferred  to  the  first 
slip  and  carefully  mixed  with  the  drop  of  water ;  from 
this  mixture  a  small  portion  is  transferred  to  the  second, 
and  from  the  second  to  the  third  slip,  and  so  on,  in  this 
way  insuring  a  dilution  of  the  number  of  organisms 
present  in  the  preparations.  These  slips  are  then  dried 
and  fixed  in  the  ordinary  way.  They  are  next  to  be 
warmed  in  the  following  solution  : 

Tannicacid  solution  in  water  (20  acid,  80  water)  ....    10  c.c. 

Cold  saturated  solution  of  ferrous  sulphate 5  c.c. 

Saturated  watery  or  alcoholic  solution  of  fuchsin    ...      1  c.c. 


METHOD  FOR  STAINING  FLAGELLA.          187 

This  solution  represents  the  mordant.     A  few  drops 
of  it  are  to  be  placed  upon  the  film  of  bacteria  on  the 

>  cover-slip,  which  is  then  to  be  held  over  a  flame  until 
the  solution  begins  to  steam.  It  should  not  be  boiled. 
After  steaming,  the  mordant  is  washed  off  in  water  and 
finally  in  alcohol.  The  bacteria  are  then  to  be  stained 

!  in  a  saturated  aniline-water-fuchsin  solution. 

When  treated  in  this  way  different  bacteria  behave 

;  differently :  the  flagella  of  some  stain  readily  in  the 
above  solutions ;  others  require  the  addition  of  an  alkali 
in  varying  quantities ;  while  others  stain  best  after  the 
addition  of  acids.  To  meet  these  conditions  an  exact 

;  1  per  cent,  solution  of  caustic  soda  in  water  must  be 

!  prepared,  and  also  a  solution  of  sulphuric  acid  in  water 
of  such  strength  that  one  cubic  centimetre  will  be  ex- 
actly neutralized  by  one  cubic  centimetre  of  the  alkaline 
solution. 

For  different  bacteria  which  have  been  studied  by 
this  method  Loffler  recommends  the  one  or  the  other  of 
these  solutions  to  be  added  to  the  mordant  in  the  follow- 
ing proportions. 
Of  the  acid  solution  : 

;   For  spirillum  cholerx  Asiaticse,  i  to  1  drop  of  acid  to  16  c.c.  of  mordant. 

For  spirillum  rubrum,  9 

'   For  spirillum  Metschnikoffi,          4  " 

For  bacillus  pyocyaneus,  5 

For  spirillum  concentricum,  no  addition  of  either  acid  or  alkali. 

Of  the  alkaline  solution  : 

For  bacillus  mesentericus  vulgatus,  4  drops  of  alkali  to  16  c.c.  of  mordant. 

For  micrococcus  agilis,  20  ' ' 

For  bacillus  typhosus,  22  " 

For  bacillus  suUilis,  28-30        "  "  "  " 

For  bacillus  cedematis  maligni,  36-37        " 

For  bacillus  anthracis  symp- } 

iomatid,  )  35 


188  BA  CTER IOL  0  G  Y. 

The  drops  used  run  22  to  the  cubic  centimetre. 

For  other  organisms  one  must  determine  whether  the 
results  are  better  after  the  addition  of  acid  or  alkali, 
and  how  much  of  either  is  required.  In  general,  it  may 
be  said  that  bacteria  which  produce  acids  in  the  media 
in  which  they  are  growing  require  the  addition  of  alka- 
lies to  the  mordant,  while  those  that  produce  alkalies 
require  acids  to  be  added.  By  following  Loftier' s  direc- 
tions the  delicate,  hair-like  flagella  on  motile  organisms 
may  be  rendered  plainly  visible. 

There  are  several  points  and  slight  modifications  in 
connection  with  this  method  that  require  to  be  empha- 
sized in  order  to  insure  success :  the  culture  to  be  em- 
ployed should  be  young,  not  over  18-20  hours  old;  it 
should  have  developed  for  this  time  on  fresh  agar-agar 
at  37°  to  38°  C. ;  the  mordant  should  not  be  perfectly 
fresh,  as  the  best  results  are  obtained  from  the  use  of 
old  solutions  that  have  stood  exposed  to  the  air  and 
that  have  been  filtered  just  before  using ;  when  placed 
on  the  cover-slip  and  held  over  the  flame  never  heat  the 
mordant  to  the  boiling-point ;  indeed,  the  best  results  are 
obtained  when  the  preparation  is  held  high  above  the  flame 
and  removed,  from  it  at  the  first  evidence  of  vaporization, 
or,  better  still,  a  little  be/ore  this  point  is  reached.  We 
have  derived  no  advantage  from  the  addition  of  acids 
or  alkalies  to  the  mordant,  as  recommended  by  Loftier ; 
but  obtain,  with  a  fair  degree  of  regularity,  satisfactory 
results  through  the  use  of  the  neutral  mordant  alone.1 

BUNGE'S  METHOD. — A  useful  modification  of  Lof- 
fler's method  is  that  recommended  by  Bunge  :  prepare 

1  I  am  indebted  to  Dr.  James  Homer  Wright,  Thomas  Scott  Fellow 
in  Hygiene,  1892-'93,  University  of  Pennsylvania,  for  some  of  the 
suggestions  in  connection  with  the  modification  of  this  method. 


DUCKWALL'S  METHOD.  189 

a  saturated  solution  of  tannin,  and  a  solution  of  liquor 
ferri  sesquichlor.  of  the  strength  of  1  :  20  of  distilled 
water.  To  3  parts  of  the  tannin  solution  add  1  part  of 
the  dilute  iron  solution.  To  10  c.c.  of  such  a  mixture 
add  1  c.c.  of  concentrated  watery  solution  of  fuchsin. 
This  mordant  is  not  to  be  used  fresh,  but  only  after 
standing  exposed  to  the  air  for  several  days  (better  for 
several  weeks).  After  preparing  the  cover-slip  with  all 
precautions  necessary  to  cleanliness  the  filtered  mordant 
is  allowed  to  act  cold  for  about  five  minutes,  after  which 
it  is  slightly  warmed ;  the  slip  is  then  washed  in 
water,  dried,  and  faintly  stained  with  carbol-fuchsin. 
No  addition  of  acid  or  alkali  to  the  mordant  is  neces- 
sary. 

DUCKWALL'S  METHOD  l  is  a  modification  of  the  Lof- 
fler  method,  and  the  results  obtained  thereby  are  very 
satisfactory. 

Preparation  of  the  Staining  Agents. — The  fixing  agent 
is  mordant,  and  the  stain  is  carbol-gentian-violet  or, 
preferably,  carbol-fuchsin. 

The  Mordant. 

Desiccated  tannic  acid 2  grammes. 

Cold  saturated  solution  ferrous  sulphate  (aqueous)      5       " 

Distilled  water 15  c.c. 

Saturated  alcoholic  solution  of  fuchsin 1   " 

The  tannic  acid  is  dissolved  in  the  water  first  by  the 
application  of  gentle  heat,  then  the  ferrous  sulphate, 
and  then  the  alcoholic  solution  of  fuchsin  are  added. 
To  these  ingredients  it  is  advisable  to  add  a  certain 
amount  of  sodium  hydroxide,  a  1  per  cent,  solution, 
varying  from  0.5  to  1  c.c.  The  best  grade  of  filter- 

1  The  Canner,  vol.  xx.  p.  23. 


190  BACTERIOLOGY. 

paper  is  used  for  filtering  the  mordant,  and  there  should 
be  left  a  heavy  precipitate.  After  filtering,  the  color 
of  this  mordant  should  be  of  a  reddish-brown  hue,  not 
clear,  but  somewhat  cloudy,  and  this  mordant  must  be 
used  within  five  hours  after  it  is  made.  After  that  time 
it  loses  its  fixing  power.  This  is  indicated  by  its 
gradual  clarification  and  darkened  color.  It  gives  the 
best  results  when  strictly  fresh,  and  accomplishes  its 
work  in  a  much  shorter  time,  so  that  very  little  if  any 
heating  is  required  when  it  is  placed  on  the  cover-glass 
preparation. 

Carbol-fuchvin  Stain.  —  Take  about  1  gramme  of 
granulated  fuchsin  (not  the  acid  fuchsin),  put  it  in  a 
bottle,  and  pour  over  it  about  25  c.c.  of  warm  absolute 
alcohol.  Shake  vigorously  and  let  it  stand  for  several 
hours  before  using.  The  carbol-fuchsin  is  made  by 
diluting  the  saturated  alcoholic  solution  four  or  five 
times  with  a  5  per  cent,  solution  of  carbolic  acid.  Car- 
bol-fuchsin should  be  freshly  made,  heated,  and  filtered 
before  using. 

The  application  of  this  method  of  demonstrating  the 
flagella  varies  with  different  organisms  with  regard  to 
the  length  of  time  the  mordant  and  stain  are  allowed  to 
act,  and  the  amount  of  sodium  hydroxide  solution  used. 
Usually,  it  is  well  to  heat  the  mordant  on  the  cover-slip 
to  steaming,  and  allow  it  to  act  from  one-half  to  one 
minute.  It  is  then  washed  off  with  water  and  a  small 
quantity  of  alcohol  poured  over  the  surface  and  washed 
off  instantly.  The  water  on  the  cover-slip  is  now 
absorbed  from  the  edge  of  the  cover-slip  with  clean 
filter-paper.  The  carbol-fuchsin  stain  is  now  applied 
and  heated  just  enough  to  generate  a  thin  vapor.  The 
stain  should  not  act  for  more  than  from  one-half  to  one 


METHOD   OF   VAN  ERMENGEM.  191 

minute.  The  cover-slip  is  now  dried,  then  xylol  is 
poured  over  the  surface,  the  excess  being  removed  with 
tilkT-paper.  The  cover-slip  is  now  mounted  in  xylol 
balsam. 

THE  METHOD  OF  VAN  ERMENGEM. — Another 
method  of  demonstrating  the  presence  of  flagella  is  that 
suggested  by  Van  Ermengem.  It  is  somewhat  more 
complicated  than  either  of  the  preceding  methods.  The 
steps  in  the  process  are  as  follows : 

In  the  centre  of  a  perfectly  cleaned  cover-slip  place 
a  drop  of  a  very  dilute  suspension,  in  physiological  salt- 
solution,  of  a  10-  to  18-hour  old  agar-agar  culture  of 
the  organism  to  be  studied.  The  suspension  of  the 
organisms  in  the  salt-solution  should  be  very  dilute  in 
order  to  favor  the  isolation  of  single  cells  on  the  slip 
and  also  to  obviate  the  occurrence  of  excessive  precip- 
itation. The  slips  are  then  to  be  dried  in  the  air  and 
fixed  over  a  gas-flame  in  the  usual  manner. 

The  mordant  used  consists  of: 

Osmic  acid  (2  per  cent,  solution) 1  part. 

Tannin  (10-25  per  cent,  solution) 2  parts. 

Place  a  drop  or  two  of  the  mordant  on  the  cover-slip 
to  be  stained,  and  allow  it  to  act  for  one-half  hour  at 
room-temperature,  or  for  five  minutes  at  50°  to  60°  C. 
Wash  carefully  in  water  and  in  alcohol,  and  then  im- 
merse for  a  few  seconds  in  the  "  sensitizing  bath,"  viz., 
a  0.25-0.5  per  cent,  solution  of  silver  nitrate.  Without 
washing,  bring  the  slip  into  a  watch-cry stalful  of  the 
"  reducing  and  reinforcing  bath/7  viz. : 

Gallic  acid 5  grammes. 

Taunin 3      " 

Fused  potassium  acetate 10      " 

Distilled  water ,                                                  .  350      " 


192  BACTERIOLOGY. 

After  a  few  seconds  pass  the  slip  back  into  a  watch- 
crystal  containing  the  dilute  silver  bath  (0.25-0.5  per 
cent,  solution  of  silver  nitrate  in  water)  and  keep  it 
in  constant  motion  until  the  solution  begins  to  take  on 
a  brown  or  blackish  color.  Wash  in  water  thoroughly  ; 
dry  with  blotting-paper,  and  mount  in  balsam. 


CHAPTER     XI. 

Systematic  study  of  an  organism — Points  to  be  considered  in  determin- 
ing the  morphologic  and  biologic  characters  of  a  culture — Methods 
by  which  the  various  biologic  and  chemical  characters  of  a  cul- 
ture may  be  ascertained — Facts  necessary  to  permit  the  identifi- 
cation of  an  organism  as  a  definite  species. 

AFTER  isolating  an  organism  in  pure  culture  by  the 
plate  method,  considerable  work  is  necessary  in  order  to 
establish  its  identity.  Small  portions  of  the  pure  cult- 
ure are  taken  upon  the  point  of  a  sterile  platinum  wire 
and  transplanted  into  the  various  culture-media.  These 
sub-cultures  of  the  organism  are  then  placed  under  suit- 
able conditions  of  temperature  and  environment,  and 
examined  from  day  to  day  to  note  the  alterations  that 
occur  in  the  different  media.  In  the  systematic  study 
of  an  organism  no  one  character  can  be  relied  upon  to 
the  exclusion  of  others.  It  is  necessary  to  note  the 
microscopic  appearance  of  the  individual  organism  and 
its  behavior  toward  different  staining  solutions  and 
other  reagents ;  in  addition  it  is  necessary  to  note  the 
gross  appearance  of  the  culture  on  the  different  media 
as  shown  by  naked-eye  (macroscopic)  examination  as 
well  as  under  a  lens  of  low  magnifying  power  (micro- 
scopic) ;  while  equal  importance  must  be  given  to  the 
chemical  alterations  produced  by  the  bacteria  in  the  dif- 
ferent media,  and  the  influence  of  different  reagents, 
when  added  to  the  media,  to  show  the  presence  of  cer- 
tain metabolic  products.  In  this  manner  the  entire  life 
history  of  an  organism,  outside  the  animal  body,  may 
be  ascertained. 

1«-  193 


194  BACTERIOLOGY. 

The  different  characters  of  an  organism  may  be 
grouped  as  :  (a)  morphologic,  those  ascertained  by  exam- 
ination of  the  individual  organism  under  a  lens  of  high 
magnifying  power;  (6)  biologic,  those  ascertained  by 
macroscopic  and  microscopic  study  of  the  gross  appear- 
ance of  the  culture  in  the  different  media;  (c)  biochemic, 
the  alterations  produced  in  the  different  media  as  shown 
by  direct  examination  or  by  the  use  of  different  reagents; 
and  (d)  pathogenic,  the  effects  of  the  inoculation  of  the 
culture  into  susceptible  animals. 

SCHEME  OF  STUDY. — Record  the  source  from  whence 
the  organism  was  derived.  Was  this  the  normal  habitat 
of  the  organism,  or  was  it  present  accidentally  ? 

MORPHOLOGIC  CHARACTERS. — Note  the  shape,  size, 
and  grouping  of  the  organism  as  it  occurs  in  the  differ- 
ent media.  Observe  the  nature  of  the  ends  of  the  indi- 
vidual organism.  Determine  the  presence  or  absence 
of  motility  in  very  young  cultures.  If  motility  is 
observed,  apply  one  of  the  special  methods  for  demon- 
strating flagella  to  note  their  relative  number  and  loca- 
tion. Stain  young  cultures  by  means  of  the  different 
staining  solutions,  and  note  the  effect  of  each.  Do  the 
organisms  stain  deeply  and  uniformly,  or  are  they 
stained  in  a  peculiar  manner  ?  Apply  the  Gram  method 
of  staining,  and  note  whether  or  not  the  organisms 
are  decolorized  by  the  alcohol.  Stain  the  organisms 
deeply  with  carbol-fuchsin  staining  solution,  and  note 
the  effect  of  different  decolorizing  agents ;  and  ascertain 
whether  the  organisms  are  capable  of  resisting  the 
decolorizing  effects  of  dilute  acids.  Do  the  organisms 
show  the  presence  of  a  capsule  when  taken  from  th( 
blood  or  tissues  of  an  animal,  or  when  taken  from  cult- 
ures in  milk  or  blood-serum  ?  Examine  cultures  thai 


BIOLOGIC  CHARACTERS.  195 

are  several  days  old,  and  note  whether  spores  are  being 
formed.  Note  particularly  the  position  of  the  spore 
within  the  cell.  Is  the  spore  of  smaller  or  greater 
diameter  than  the  cell  in  which  it  is  forming  ?  Exam- 
ine cultures  that  are  a  week  or  more  old,  and  note 
whether  the  organisms  have  undergone  any  definite 
alterations  in  form  (involution  forms),  or  whether  they 
present  evidences  of  fragmentation  or  granulation  of 
their  protoplasm  (degeneration  forms). 

BIOLOGIC  CHARACTERS.  —  Colony-formation.  —  Ob- 
serve the  character  of  the  colonies  formed  in  gelatin 
and  agar-agar  plates.  Describe  a  typical  surface  colony 
and  a  typical  deep  colony,  both  as  to  their  macroscopic 
and  microscopic  appearance.  What  is  the  relative  size 
of  the  colonies  formed  on  each  of  these  media  when 
they  are  sufficiently  separated  from  one  another  to  permit 
unhindered  development  ?  Note  the  color  and  internal 
structure  of  the  colonies  as  well  as  their  relative  density. 
What  is  the  nature  of  the  surface  contour  and  arrange- 
ment of  the  colonies  ?  Note  their  general  character,  as 
to  whether  they  are  moist  or  dry,  compact  or  loosely 
constructed,  sharply  circumscribed  or  spreading  over  the 
surface  of  the  medium.  Do  the  gelatin  colonies  show 
evidences  of  liquefaction  ? 

Agar-slant  Inoculations. — Observe  the  nature  of  the 
growth  on  the  surface  of  an  agar-agar  slant  inoculation. 
Describe  the  color,  texture,  and  optical  characters  of  the 
growth.  Is  the  growth  confined  to  the  line  of  inocula- 
tion, or  has  it  a  tendency  to  spread  over  the  surface  of 
the  medium  ?  Is  it  smooth  or  rough,  moist  or  dry, 
glistening  or  dull  in  character?  If  the  organism  forms 
pigment,  note  whether  the  pigment  is  confined  to  the 
area  of  growth  or  whether  it  extends  into  the  medium 


196  BACTERIOLOGY. 

itself.  Record  the  manner  in  which  the  culture  changes 
in  its  appearance  on  successive  days. 

Agar-stab  Inoculations. — Observe  the  nature  of  the 
growth  in  an  agar-agar-stab  inoculation.  Note  whether 
the  growth  is  most  voluminous  at  or  near  the  surface 
or  in  the  depth  of  the  stab.  If  the  organism  produces 
pigment,  note  whether  the  pigment-formation  is  most 
marked  at  or  near  the  surface  or  at  the  bottom  of  the 
stab.  Record  the  alterations  that  are  observed  on  sev- 
eral successive  days. 

Gelatin-stab  Inoculations. — Observe  the  nature  of  the 
growth  in  a  gelatin-stab  inoculation.  Is  the  growth 
most  voluminous  at  or  near  the  surface  or  at  the  bottom 
of  the  stab  ?  Note  the  general  character  of  the  growth 
on  the  surface,  especially  as  to  its  contour,  extent,  and 
color.  Note  the  character  of  the  growth  in  the  stab. 
Is  it  continuous  along  the  whole  line  of  inoculation,  or 
is  it  confined  to  isolated  areas  ?  If  the  organism  has 
the  property  of  liquefying  gelatin,  note  carefully  the 
manner  in  which  the  liquefaction  proceeds.  How  soon 
does  liquefaction  begin,  and  in  what  length  of  time  is  a 
tube  of  gelatin  completely  liquefied? 

Potato  Culture. — Observe  the  nature  of  the  growth  on 
potato.  This  is  an  important  differentiating  medium, 
since  some  organisms  grow  very  sparingly  or  without 
producing  a  visible  growth.  Other  organisms  grow 
very  characteristically.  Some  organisms  have  the  prop- 
erty of  breaking  up  the  starch  of  the  potato  into  sim- 
pler compounds.  This  is  sometimes  manifested  by  the 
formation  of  gas.  Many  of  the  chromogenic  bacteria 
find  the  potato  a  most  suitable  pabulum  on  which  to 
form  their  pigment,  the  pigment  formed  on  this  medium 
having  at  times  an  especial  brilliancy.  Note  in  detail 


BIOLOGIC  CHARACTERS.  197 

all  the  changes  that  occur  in  the  growth  on  successive 
days. 

Growth  in  Bouillon. — Observe  the  nature  of  the 
growth  in  bouillon.  Note  whether  the  fluid  shows  tur- 
bidity or  not,  as  well  as  the  extent  and  distribution  of 
this  alteration.  Note  whether  any  sediment  is  being 
formed,  as  well  as  the  nature  and  amount  of  such  sedi- 
ment. Does  the  organism  form  a  definite  growth  (pel- 
licle or  scum)  on  the  surface  of  the  bouillon  ?  What  is 
the  character  of  the  pellicle  ?  Is  is  readily  dislodged, 
and,  when  dislodged,  is  it  replaced  by  a  new  pellicle  ? 
Note  whether  the  color  of  the  medium  has  become 
altered.  Note  the  manner  in  which  the  appearance  of 
the  culture  changes  on  several  successive  days. 

Growth  in  Litmus-milk. — Observe  the  nature  of  the 
growth  in  litmus-milk.  Has  the  reaction  of  the  medium 
become  altered?  To  what  is  such  alteration  attribu- 
table? Note  whether  there  is  precipitation  of  casein. 
Record  the  extent  and  rapidity  with  which  this  altera- 
tion takes  place,  as  well  as  the  reaction  of  the  fluid 
while  the  change  is  being  produced.  Is  there  any  evi- 
dence of  the  subsequent  liquefaction  of  the  precipitated 
casein  ?  Has  the  litmus  been  altered  in  any  manner 
except  as  shown  by  altered  reaction  of  the  medium? 
In  what  part  of  the  tube  has  such  alteration  of  the 
litmus  commenced?  If  the  litmus  has  been  decolorized, 
is  it  possible  to  restore  its  color  by  the  admixture  of  air 
with  the  fluid?  Note  the  manner  in  which  the  appear- 
ance of  the  medium  changes  on  successive  days. 

Growth  in  Special  Media. — The  special  culture-media 
may  be  employed  to  ascertain  additional  biologic  char- 
acters of  an  organism,  such  as  the  production  of  indol, 
reduction  of  nitrates  to  nitrites,  the  formation  of  ammo- 


198  BACTERIOLOGY. 

ma,  production  of  gas  in  media  containing  different  car- 
bohydrates, or  the  reducing  power  of  the  organism  on 
aniline  dyes,  etc. 

Influence  of  External  Agencies. — Note  the  vitality  of 
the  organism  under  the  influence  of  various  physical  and 
chemical  agents.  Determine  the  temperature  at  which 
it  thrives  best,  as  well  as  the  lowest  and  highest  tem- 
peratures at  which  growth  is  possible.  Determine  the 
thermal  death-point  of  the  organism  by  subjecting  it  to 
various  degrees  of  temperature  from  55°  to  75°  0.  for 
ten  minutes.  Determine  its  resistance  to  drying ;  to 
the  influence  of  light ;  to  the  influence  of  germicidal 
substances.  Determine  the  influence  of  different  gases 
upon  the  growth  of  the  organism,  such  as  hydrogen, 
nitrogen,  or  carbon  dioxide.  Determine  the  chemical 
reaction  of  the  culture-media  best  adapted  for  its 
growth.1 

BIOCHEMIC  CHARACTERS. — If  the  organism  exhibits 
chromogenic  properties,  ascertain  whether  the  pigment 
is  intra-  or  extracellular.  Ascertain  under  what  condi- 
tions of  temperature,  reaction,  and  constitution  of  media, 
or  under  what  atmospheric  conditions  this  function  is 
best  exhibited.  Note  the  influence  of  different  reagents 
upon  the  pigment,  such  as  chloroform,  ether,  alcohol, 
water,  acids,  or  alkalies.  Note  whether  the  organism 
exhibits  photogenic  properties,  and  if  so,  ascertain  what 
conditions  are  most  suitable  for  the  manifestation  of  this 
phenomenon. 

Ascertain  whether  or  not  the  organism  produces  en- 
zymes. Does  it  manifest  a  proteolytic  function,  as  shown 

1  For  more  detailed  description  of  the  variations  in  the  character 
of  the  macroscopic  and  microscopic  appearance  of  the  cultures  in  the 
different  media,  the  student  is  referred  to  Chester  s  Determinative 
Bacteriology  and  Eyre's  Bacteriologic  Technique. 


BIOCHEMIC  CHARACTERS.  199 

by  the  liquefaction  of  gelatin,  casein,  or  blood-serum  ? 
Note  whether  this  function  is  manifested  in  alkaline  or 
in  acid  condition  of  the  medium.  Does  it  manifest  a 
precipitating  effect  (rennet  ferment?)  upon  casein? 
Note  whether  this  is  manifested  in  alkaline  or  in  acid 
condition  of  the  medium.  Does  the  organism  have  the 
property  of  breaking  up  any  of  the  carbohydrates  into 
simpler  compounds?  Is  this  alteration  accompanied  or 
not  by  the  liberation  of  gas  ?  If  so,  ascertain  the  rela- 
tive amount  of  gas  formed  from  a  given  quantity  of 
carbohydrate.  Analyze  the  gas  formed,  and  state  the 
relative  proportion  of  carbon  dioxide  and  residual  (ex- 
plosive) gas  formed.  If  the  carbohydrates  are  broken 
up  without  the  evolution  of  gas,  then  ascertain  what 
intermediary  and  end-products  are  formed.  Are  acids, 
aldehyde,  or  alcohol  formed?  Ascertain  the  nature 
of  the  acids  produced.  If  lactic  acid  is  formed 
from  lactose,  ascertain  its  character  by  means  of  the 
polariscope. 

Ascertain  whether  the  organism  produces  indol, 
phenol,  or  skatol.  Are  these  substances  formed  with 
the  simultaneous  reduction  of  nitrates  to  nitrites?  Are 
the  nitrites  reduced  further  into  ammonia  ? 

Pathogenic  Properties. — Ascertain  whether  any  of  the 
animals  used  for  experimental  purposes  are  susceptible 
when  inoculated  with  the  organism.  Are  all  species  of 
laboratory  animals  equally  susceptible,  or  are  some 
immune?  Note  the  size  of  the  dose  and  the  manner 
of  inoculation  that  gives  the  most  constant  and  charac- 
toristie  results.  What  are  the  symptoms  and  post- 
mortem appearances  produced  ?  What  is  the  location 
of  the  organisms  in  the  body  of  the  dead  animal  ?  Are 
they  confined  to  the  seat  of  inoculation  (toxemia),  or  are 


200  BACTERIOLOGY. 

they  distributed  more  or  less  generally  throughout  the 
body  (bacteruemia)  ? 

Note  whether  the  virulence  of  the  organism  is  main- 
tained when  grown  for  several  generations  on  artificial 
media,  or  whether  it  soon  becomes  attenuated.  Which 
culture-medium  is  best  suited  to  conserve  the  virulence 
of  the  organism  ?  In  what  manner  does  its  environ- 
ment influence  the  virulence  ?  If  the  virulence  is  readily 
lost,  may  it  be  regained  by  any  of  the  known  methods? 

Ascertain  whether  the  organism  forms  a  soluble  toxin 
when  grown  in  fluid  media,  as  sugar-free  bouillon.  If 
toxin  is  formed,  ascertain  whether  the  antitoxic  state  is 
readily  induced  in  susceptible  animals. 

If  no  soluble  toxin  is  formed,  ascertain  whether  ani- 
mals may  be  immunized  by  the  injection  of  sub-lethal 
doses  of  dead  or  living  cultures.  Is  a  bactericidal  immu- 
nity induced  by  this  means?  Does  the  serum  of  immune 
animals  possess  protective  and  curative  properties  when 
administered  to  susceptible  animals  before  or  after  inoc- 
ulation with  the  living  organism  ?  Does  the  serum  of 
immune  animals  possess  the  property  of  agglutinating 
the  organisms  in  relatively  higher  dilutions  than  the 
serum  of  normal  animals  of  the  same  species  ? 

The  majority  of  the  bacteria  may  be  identified  with- 
out resorting  to  such  a  detailed  study  of  the  biochemic 
and  pathogenic  properties  as  given  in  the  foregoing  out- 
line, but  for  some  of  the  pathogenic  bacteria  it  has  been 
necessary  to  apply  all  the  known  tests  in  order  to  defi- 
nitely establish  their  identity.  By  means  of  such 
detailed  studies  on  related  organisms,  it  has  been  possi- 
ble to  differentiate  varieties  whose  characters  are  con- 
stant, yet  in  general  they  are  so  closely  related  that  it  is 
impossible  from  the  clinical  manifestations  produced  to 


DIFFERENT  PARTS  OF  THE  MICROSCOPE.    201 

state  definitely  which  particular  variety  of  organism  is 
responsible  for  the  conditions.  This  is  especially  true 
of  the  different  varieties  of  bacillus  dysenteria?,  and  of 
the  group  of  typhoid  and  paratyphoid  organisms.  Fur- 
ther study  will,  no  doubt,  reveal  variations  in  other 
pathogenic  bacteria,  which  varieties  are  to-day  regarded 
as  a  distinct  species. 

MICROSCOPIC  EXAMINATION  OF  PREPARATIONS. 

THE  DIFFERENT  PARTS  OF  THE  MICROSCOPE. — 
Before  describing  the  method  of  examining  prepara- 
tions microscopically,  a  few  definitions  of  the  terms 
used  in  connection  with  the  microscope  may  not  be  out 
of  place.  (The  different  parts  of  the  microscope  referred 
to  below  are  indicated  by  letters  in  Fig.  35.) 

The  ocular  or  eye-piece  (A)  is  the  lens  at  which  the 
eye  is  placed  when  looking  through  the  instrument.  It 
serves  to  magnify  the  image  projected  through  the  ob- 
jective. 

The  objective  (B)  is  the  lens  which  is  at  the  distal 
end  of  the  barrel  of  the  instrument,  and  which  serves 
to  magnify  the  object  to  be  examined. 

The  stage  (c)  is  the  shelf  or  platform  of  the  micro- 
scope on  which  the  object  to  be  examined  rests. 

The  diaphragms  are  the  perforated  stops  that  fit  in 
the  centre  of  the  stage.  They  vary  in  size,  so  that  dif- 
ferent amounts  of  light  may  be  admitted  to  the  object 
by  using  diaphragms  with  larger  or  smaller  openings. 

The  "  -iris  "  diaphragm  (D)  opens  and  closes  like  the 
iris  of  the  eye.  It  is  so  arranged  that  its  opening  for 
admission  of  light  can  be  increased  or  diminished  by 
moving  a  small  lever  in  one  or  another  direction. 

The    reflector   (E)  is  the  mirror  placed   beneath  the 


202 


BACTERIOLOGY. 


stage,  which  serves  to  illuminate  the  object  to  be  exam- 
ined. 

The  coarse  adjustment  (F)  is  the  rack-and-pinion  ar- 


FlG.  35. 


— G 


rangement  by  which  the  barrel  of  the  microscope  can 
be  quickly  raised  or  lowered. 


DIFFERENT  PARTS  OF  THE  MICROSCOPE.    203 

The  fine  adjustment  (G)  serves  to  raise  and  lower  the 
barrel  of  the  instrument  very  slowly  and  gradually. 

For  the  microscopic  study  of  bacteria  it  is  essential 
that  the  microscope  be  provided  with  an  oil-immersion 
system  and  a  sub-stage  condensing  apparatus. 

The  oil-immersion  or  homogeneous  system  consists  of 
an  objective  so  constructed  that  it  can  only  be  used  when 
the  transparent  media  through  which  the  light  passes  in 
entering  it  are  all  of  the  same  index  of  refraction — i.  e., 
are  homogeneous.  This  is  accomplished  by  interposing 
between  the  face  of  the  lens  and  the  cover-slip  covering 
the  object  to  be  examined  a  body  which  refracts  the 
light  in  the  same  way  as  do  the  glass  slide,  the  cover- 
slip,  and  the  glass  of  which  the  objective  is  made.  For 
this  purpose,  a  drop  of  oil  of  the  same  index  of  refrac- 
tion as  the  glass  is  placed  upon  the  face  of  the  lens, 
and  the  examinations  are  made  through  this  oil.  There 
is  thus  little  or  no  loss  of  light  from  deflection,  as  is  the 
case  in  the  dry  system. 

The  sub-stage  condensing  apparatus  (H)  is  a  system 
of  lenses  situated  beneath  the  central  opening  of  the 
stage.  They  serve  to  condense  the  light  passing  from 
the  reflector  to  the  object  in  such  a  way  that  it  is 
focussed  upon  the  object,  thus  furnishing  the  greatest 
amount  of  illumination.  Between  the  condenser  and 
reflector  is  placed  the  "  iris  "  diaphragm,  the  aperture 
of  which  can  be  regulated,  as  circumstances  require,  to 
permit  of  either  a  very  small  or  a  very  large  amount  of 
light  passing  to  the  object. 

The  nose-piece  (i)  consists  of  a  collar,  or  group  of 
collars  joined  together  (two  or  more),  that  is  attached  to 
the  distal  end  of  the  tube  of  the  microscope.  It  enables 
one  to  attach  several  objectives  to  the  instrument  in 


204  BACTERIOLOGY. 

such  a  way  that  by  simply  rotating  the  nose-piece  the 
various  lenses  of  different  power  may  be  conveniently 
used  in  succession. 

MICROSCOPIC  EXAMINATION  OF  COVER-SUPS. — The 
stained  cover-slip  is  to  be  examined  with  the  oil-immer- 
sion objective,  and  with  the  diaphragm  of  the  sub-stage 
condensing  apparatus  open  to  its  full  extent.  The  object 
gained  by  allowing  the  light  to  enter  in  such  a  large  vol- 
ume is  that  the  contrast  produced  by  the  colored  bacteria 
in  the  brightly  illuminated  field  is  much  more  conspicu- 
ous than  when  a  smaller  amount  of  light  is  thrown  upon 
them.  This  is  true  not  only  for  stained  bacteria  on 
cover-slips,  but  likewise  for  their  differentiation  from 
surrounding  objects  when  they  are  located  in  tissues. 
With  unstained  bacteria  and  tissues,  on  the  contrary,  the 
structure  can  best  be  made  out  by  reducing  the  bundle 
of  light-rays  to  the  smallest  amount  compatible  with 
distinct  vision,  and  in  this  way  favoring,  not  color-con- 
trast, but  contrasts  which  appear  as  lights  and  shadows, 
due  to  the  differences  in  permeability  to  light  of  the 
various  parts  of  the  material  under  examination. 

STEPS  IN  EXAMINING  STAINED  PREPARATIONS 
WITH  THE  OIL-IMMERSION  SYSTEM. — Place  upon  the 
centre  of  the  cover-slip  which  covers  the  preparation  a 
small  drop  of  immersion  oil.  Place  the  slide  upon  the 
centre  of  the  stage  of  the  microscope.  With  the  coarse 
adjustment  lower  the  oil-immersion  objective  until  it 
j-ust  touches  the  drop  of  oil.  Open  the  illuminating 
apparatus  to  its  full  extent.  Then,  with  the  eye  to  the 
ocular  and  the  hand  on  the  fine  adjustment,  turn  the 
adjusting-screw  toward  the  right  until  the  field  becomes 
somewhat  colored  in  appearance.  When  this  is  seen 
proceed  more  slowly  in  the  same  direction,  and,  after 


UNSTAINED  PREPARATIONS.  205 

one  or  two  turns,  the  object  will  be  in  focus.  Do  not 
remove  the  eye  from  the  instrument  until  this  has  been 
accomplished. 

Then,  with  one  hand  upon  the  fine  adjustment  and 
the  thumb  and  index  finger  of  the  other  hand  hold- 
ing the  slide  lightly  by  its  end,  it  may  be  moved  about 
under  the  objective.  At  the  same  time  the  screw 
of  the  fine  adjustment  must  be  turned  back  and  forth, 
so  that  the  different  planes  of  the  preparation  may  be 
brought  into  focus  one  after  the  other.  In  this  way  the 
whole  section  or  preparation  may  be  inspected.  When 
the  examination  is  finished  raise  the  objective  from  the 
preparation  by  turning  the  screw  of  the  coarse  adjust- 
ment toward  you.  Remove  the  preparation  from  the 
stage,  and,  with  a  fine  silk  cloth  or  handkerchief,  wipe 
very  gently  and  carefully  the  oil  from  the  face  of  the  lens. 
The  lens  is  then  unscrewed  from  the  microscope  and 
placed  in  the  case  intended  for  its  reception. 

During  work,  of  course,  the  lens  need  not  be  cleaned 
and  put  away  after  each  examination ;  but  when  the 
work  for  the  day  is  over  an  immersion  lens  must 
always  be  protected  in  this  way.  Under  no  circum- 
stances should  it  be  allowed  to  remain  in  the  immersion 
oil  or  exposed  to  dust  for  any  length  of  time. 

EXAMINATION  OF  UNSTAINED  PREPARATIONS.— 
"  Hanging  drops."  It  frequently  becomes  necessary  to 
examine  bacteria  in  the  unstained  condition. .  The  cir- 
cumstances calling  for  this  arise  while  studying  the 
multiplication  of  cells,  the  germination  of  spores,  the 
formation  of  spores,  and  the  absence  or  presence  of 
motility. 

In  this  method  the  organisms  to  be  studied  are  sus- 
pended in  a  drop  of  physiological  salt-solution  or  bou- 


20  6  BA  CTERIOL  OGY. 

illon  in  the  centre  of  a  cover-slip.  This  is  then  placed, 
drop  down,  upon  a  slide,  in  the  centre  of  which  a  hollow 
or  depression  is  ground  (Fig.  36).  The  slip  is  held  in 


FIG.  36. 


Longitudinal  section  of  hollow-ground  glass  slide  for  observing  bacteria  in 
hanging  drops. 

position  by  a  thin  layer  of  vaselin,  which  may  be 
painted  around  the  margins  of  the  depression.  This 
not  only  prevents  the  slip  changing  its  position  during 
examination,  but  also  prevents  drying  by  evaporation 
if  the  preparation  is  to  be  observed  for  any  length 
of  time.  This  is  known  as  the  "hanging-drop" 
method  of  examination  or  cultivation.  It  is  indispen- 
sable for  the  purposes  mentioned,  and  at  the  same  time 
requires  considerable  care  in  its  manipulation.  The 
fluid  is  so  transparent  that  the  cover-slip  is  often  broken 
by  the  objective  being  brought  down  upon  the  prepara- 
tion before  one  is  aware  that  the  focal  distance  has  been 
reached.  This  may  be  avoided  by  grasping  the  slide 
with  the  left  hand  and  moving  it  back  and  forth  under 
the  objective  as  it  is  moved  toward  the  object.  As  soon 
as  the  least  pressure  is  felt  upon  the  slide  the  objective 
must  be  raised,  otherwise  the  cover-slip  will  be  broken 
and  the  lens  may  be  rendered  worthless. 

A  safer  plan  is  to  bring  the  edge  of  the  drop  into  the 
centre  of  the  field  with  one  of  the  higher  power  dry 
lenses.  When  this  is  accomplished  substitute  the  im- 
mersion for  the  dry  system,  when  the  edge  of  the  drop 
is  readily  detected  with  the  higher  power  lens  some- 
where near  the  centre  of  the  field. 

In  examining  bacteria  by  this  method  there  is  a  pos- 


STUDY  OF  SPORE-FORMATION.  207 

sibility  of  error  that  must  be  guarded  against.  All 
microscopic  insoluble  particles  in  suspension  in  fluids 
possess  a  peculiar  tremor  or  vibratory  motion,  the  so- 
called  "  Brownian  motion."  This  is  very  apt  to  give 
the  impression  that  the  organisms  under  examination 
are  motile,  when  in  truth  they  are  not  so,  their  move- 
ment in  the  fluid  being  only  this  molecular  tremor. 

The  difference  between  the  motion  of  bodies  under- 
going this  molecular  tremor  and  that  possessed  by  cer- 
tain living  bacteria  is  that  the  former  particles  never 
move  from  their  place  in  the  field,  while  living  bac- 
teria alter  their  position  in  relation  to  the  surround- 
ing organisms,  and  may  dart  from  one  position  in  the 
I  field  to  another.  In  some  cases  the  true  movement  of 
bacteria  is  very  slow  and  undulating,  while  in  others  it 
is  rapid  and  darting.  The  molecular  tremor  may  be 
seen  with  non-motile  and  with  dead  organisms. 

NOTE. — Prepare  three  hanging-drop  preparations — 
one  from  a  drop  of  dilute  India-ink,  a  second  from  a 
culture  of  micrococci,  and  a  third  from  a  culture  of  the 
bacillus  of  typhoid  fever.  In  what  way  do  they  differ  ? 

STUDY  OF  SPORE-FORMATION. — The  hanging-drop 
method  just  mentioned  is  not  only  employed  for  detect- 
ing the  motility  of  an  organism,  but  also  for  the  study 
of  its  mode  of  spore-formation. 

Since  with  aerobic  organisms  spore-formation  occurs, 
as  a  rule,  only  in  the  presence  of  oxygen,  and  is  induced 
more  by  limitation  of  the  nutrition  of  the  organisms 
than  by  any  other  factor,  it  is  essential  that  these  two 
points  should  be  borne  in  rnind  in  preparing  the  drop- 
cultures  in  which  the  process  is  to  be  studied.  For  this 


208  BACTERIOLOGY. 

reason  the  drop  of  bouillon  should  be  small  and  the 
air-chamber  relatively  large. 

The  cover-slip  and  hollow-ground  slide  should  be 
carefully  sterilized,  and  with  a  sterilized  platinum  loop 
a  very  small  drop  of  bouillon  is  placed  in  the  centre 
of  the  cover-slip.  The  slip  is  then  inverted  over 
the  hollow  depression  in  the  sterilized  object-glass  and 
sealed  with  vaselin.  The  most  convenient  method  of 
performing  this  last  step  in  the  process  is  to  paint  a 
ring  of  vaselin  around  the  edges  of  the  hollow  in  the 
slide,  and  then,  without  taking  the  cover-slip  from  the 
table  upon  which  it  rests,  invert  the  hollow  over  the 
drop  and  press  it  gently  down  upon  the  cover-slip.  The 
vaselin  causes  the  slip  to  adhere  to  the  slide,  so  that  it 
can  be  easily  taken  up.  The  drop  now  hangs  in  the 
centre  of  the  small  air-tight  chamber  which  exists  be- 
tween the  depression  in  the  slide  and  the  cover-slip. 
(See  Fig.  36.) 

A  very  thin  drop  of  sterilized  agar-agar  may  be  sub- 
stituted for  the  bouillon.  It  serves  to  retain  the  organ- 
isms in  a  fixed  position,  and  the  process  may  be  more 
easily  followed. 

As  soon  as  finished  the  preparation  is  to  be  examined 
microscopically  and  the  condition  of  the  organisms 
noted.  It  is  then  to  be  retained  in  a  warm  chamber 
especially  devised  for  the  purpose,  and  kept  under  con- 
tinuous observation.  The  form  of  chamber  best  adapted 
to  the  purpose  is  one  which  envelops  the  whole  micro- 
scope. It  is  provided  with  a  window  through  •  which 
the  light  enters,  and  an  arrangement  by  which  the  slide 
may  be  moved  from  the  outside.  The  formation  of 
spores  requires  a  much  longer  time  than  the  germina- 


STUDY  OF  SPORE-FORMATION.  209 

tion  of  spores  into  bacilli,  but  with  patience  both  proc- 
esses may  be  satisfactorily  observed. 

It  will  be  noticed  that  the  description  of  this  process 
is  very  much  like  that  which  immediately  precedes,  but 
differs  from  it  in  one  respect,  viz.,  that  in  this  manipu- 
lation we  are  not  making  a  preparation  which  is  simply 
to  be  examined  and  then  thrown  aside,  but  it  is  an 
actual  pure  culture,  and  must  be  kept  as  such,  otherwise 
the  observation  will  be  worthless.  For  this  reason  the 
greatest  care  must  be  observed  in  the  sterilization  of 
till  objects  employed.  Studies  upon  spore-formation  by 
this  method  frequently  continue  over  hours,  and  some- 
times days,  and  contamination  must,  therefore,  be  care- 
fully guarded  against.  The  study  should  be  begun  with 
the  vegetative  form  of  the  organisms ;  the  hanging-drop 
preparation  should,  for  this  reason,  always  be  made 
from  a  perfectly  fresh  culture  of  the  organism  under 
consideration  before  time  has  elapsed  for  spores  to  form. 

The  simple  detection  of  the  presence  or  absence  of 
spore-formation  can  in  many  cases  be  made  by  other 
methods.  For  example,  many  species  of  bacteria  which 
possess  this  property  form  spores  most  readily  upon 
media  from  which  it  is  sojnewhat  difficult  for  them  to 
obtain  the  necessary  nourishment ;  potatoes  and  agar-agar 
that  have  become  a  little  dry  oifer  very  favorable  con- 
ditions, because  of  the  limited  area  from  which  the 
i  growing  bacteria  can  draw  their  nutritive  supplies,  and 
i  because  of  the  free  access  which  they  have  to  oxygen, 
for,  their  growth  being  on  the  surface,  they  are  sur- 
rounded by  this  gas  unless  means  are  taken  to  prevent 
it.  By  the  hanging-drop  method,  however,  more  than 
this  specific  property  may  be  determined.  It  is  possible 
not  only  to  detect  the  stages  and  steps  in  the  formation 
14 


210  BA  CTERIOL  OGY. 

of  endogenous  spores,  but  when  the  spores  are  com- 
pletely formed  their  germination  into  mature  rods  may 
be  seen  by  transferring  them  to  a  fresh  bouillon-drop 
or  drop  of  agar-agar  preserved  in  the  same  way.  The 
word  rods  is  used  because  we  have  as  yet  no  evidence 
that  endogenous  spore-formation  occurs  in  any  of  the 
other  morphological  groups  of  bacteria. 

HANGING-BLOCK  CULTURES. — Hill1  has  devised  a 
method  for  observing  the  development  of  individual 
bacteria,  which  consists  in  the  substitution  for  the  ordi- 
nary "  hanging  drop  "  of  liquid  or  jelly  a  cube  of  solid- 
ified agar-agar,  on  the  surface  of  which  the  bacteria  are 
distributed. 

The  "  hanging  block  "  is  prepared  as  follows  :  "  Pour 
melted  nutrient  agar  into  a  Petri  dish  to  the  depth  of 
one-eighth  to  one-quarter  inch.  Cool  this  agar  and  cut 
from  it  a  block  about  one-quarter  to  one-third  inch 
square  and  of  the  thickness  of  the  layer  of  agar  in  the 
dish.  This  block  has  a  smooth  upper  and  under  sur- 
face. Place  it,  under  surface  down,  on  a  slide  and 
protect  it  from  dust.  Prepare  an  emulsion  in  sterile 
water  of  the  organism  to  be  examined  if  it  has  been 
grown  on  a  solid  medium,  or  use  a  broth  culture ;  spread 
the  emulsion  or  broth  upon  the  upper  surface  of  the 
block,  as  if  making  an  ordinary  cover-slip  preparation. 
Keep  the  slide  and  block  in  an  incubator  at  37°  C.  for  five 
to  ten  minutes  to  dry  slightly.  Then  lay  a  clean  sterile 
cover-slip  on  the  inoculated  surface  of  the  block  in 
close  contact  with  it,  avoiding,  if  possible,  the  forma- 
tion of  air-bubbles.  Remove  the  slide  from  the  lower 
surface  of  the  block,  and  invert  the  cover-slip  so  that  the 
agar  block  is  uppermost.  With  a  platinum  loop  run  a 

1  Hill:  Journal  of  Medical  Research,  vol.  vii.,  1902,  p.  202. 


CULTIVATION  IN  COLLODION  CAPSULES.      211 

drop  or  two  of  melted  agar  along  each  side  of  the  agar 
block  where  it  is  in  contact  with  the  cover-slip.  This 
seal  hardens  at  once,  preventing  slipping  of  the  block. 
Place  the  preparation  in  the  incubator  again  for 
five  or  ten  minutes  to  dry  the  agar  seal.  Invert  this 
preparation  over  a  moist  chamber  and  seal  the  cover- 
slip  in  place  with  white  wax  or  paraffin.  Yaselin 
softens  too  readily  at  37°  C.,  allowing  shifting  of  the 

;  cover-slip.     The  preparation  may  then  be  examined  at 

\  leisure." 

Aerobic   bacteria   receive    sufficient   oxygen    by  dif- 

I  fusion,    and    for   anaerobic    bacteria    it    will    suffice    to 

I  expose  the  block   to   the  action  of  alkaline  pyrogallic 

!  solution. 

CULTIVATION  OF  BACTERIA  IN  COLLODION  CAP- 

.SULES. — The  use  of  small  capsules  of  collodion  for  the 
cultivation  of  bacteria  was  first  introduced  by  Metschni- 
koff,  Roux,  and  Salembini.  The  bacteria  under  study  are 
placed  in  a  small  collodion  capsule  containing  a  nutritive 
fluid,  as  bouillon,  and,  after  sealing  the  capsule,  it  is 
placed  in  the  peritoneal  cavity  of  an  animal.  Under 
these  conditions  the  bacteria  are  protected  against  the 
phagocytic  action  of  the  body-cells,  though  the  metabolic 
products  of  the  bacteria  diffuse  out  and  act  upon  the 
animal. 

Collodion  capsules  are  easily  constructed  by  the  method 
(of  McCrae,  as  follows  :  Fuse  a  short  piece  of  narrow, 
'thin-walled  glass  tubing  into  the  top  of  a  No.  12  gelatin 
capsule.  This  is  done  by  simply  heating  one  end  of  the 
jlass  tubing  in  the  flame  of  a  Bunsen  burner  and  push- 
ng  it  through  the  top  of  the  capsule.  Both  halves  of  the 
'j^latiri  capsule  are  now  fitted  together,  and  the  closed 
bapsule  is  dipped  repeatedly  into  liquid  collodion.  When 


212  BACTERIOLOGY. 

the  covering  of  collodion  on  the  exterior  of  the  capsule 
is  of  sufficient  thickness — /.  e.,  about  that  of  writing 
paper — it  is  allowed  to  dry  for  a  short  time,  and  is  then 
filled  with  water  and  placed  in  a  pan  of  hot  water.  This 
will  dissolve  out  the  gelatin  frame-work  and  leave  the 
collodion  envelope.  The  water  is  now  removed  from 
the  capsule  by  means  of  a  capillary  pipette.  The  nutri- 
tive fluid  is  introduced  by  the  same  means,  and  the  filled 
capsule  is  dropped  into  a  tube  of  bouillon  and  sterilized, 
after  which  the  fluid  is  inoculated  and  the  glass  tube  is 
sealed  off  in  the  gas-flame.  Before  using  the  inoculated 
capsule  its  tightness  must  be  tested.  This  is  done  by 
dropping  it  into  a  tube  of  sterile  bouillon  and  placing  in 
the  incubator  for  twenty-four  hours.  If  growth  appears 
in  the  bouillon  of  the  tube,  then  either  the  wall  of  the  cap- 
sule is  permeable  to  bacteria,  or  the  bouillon  has  been  con- 
taminated during  the  manipulation.  As  the  wall  of  the 
capsule  is  to  possess  the  physical  qualities  of  an  osmotic 
membrane,  through  which  only  dialyzable  substances  can 
pass,  it  must,  naturally,  be  free  from  cracks  or  air-holes, 
and  in  making  the  capsules  the  greatest  care  must  be 
taken  to  eliminate  these  defects. 

STUDY  OF  GELATIN  CULTURES. — As  has  been  pre- 
viously stated,  the  behavior  of  bacteria  towrard  gelatin 
differs — some  of  them  producing  apparently  no  altera- 
tion in  the  medium,  while  the  growth  of  others  is 
accompanied  by  an  enzymotic  action  that  results  in 
liquefaction  of  the  gelatin  at  and  around  the  place  at 
which  the  colonies  are  growing.  In  some  instances 
this  liquefaction  spreads  laterally  and  downward,  caus- 
ing a  saucer-shaped  excavation  ;  while  in  others  the 
colony  sinks  almost  vertically  into  the  gelatin  and  may 
be  seen  lying  at  the  bottom  of  a  funnel-shaped  depres- 


CULTURES  ON  POTATO.  213 

si  on.  These  differences  are  constantly  employed  as 
one  of  the  means  of  differentiating  otherwise  closely 
allied  members  of  the  same  family  of  bacteria.  (See 
Fig.  33.)  Studies  upon  the  spirillum  of  Asiatic 
cholera  and  a  number  of  closely  allied  species,  for 
example,  reveal  decided  differences  in  the  form  of 
liquefaction  produced  by  these  various  organisms. 
The  minutest  detail  in  this  respect  must  be  noted,  and 
its  frequency  or  constancy  under  varying  conditions 
determined. 

CULTURES  ON  POTATO. — A  very  important  feature 
in  the  study  of  an  organism  is  its  growth  on  sterilized 
potato.  Many  organisms  present  appearances  under 
this  method  of  cultivation  which  alone  can  almost  be 
considered  characteristic.  In  some  cases  coarsely  lob- 
ulated,  elevated,  dry  or  moist  patches  of  development 
occur  after  a  few  hours ;  again,  the  growth  may  be  finely 
granular  and  but  slightly  elevated  above  the  surface  of 
the  potato;  at  one  time  it  will  be  dry  and  dull  in  ap- 
pearance, again  it  may  be  moist  and  glistening.  Some- 
times bubbles,  due  to  the  fermentative  action  of  the 
growing  bacteria  on  the  carbohydrates  of  the  potato, 
are  produced. 

A  most  striking  form  of  development  on  potato  is  that 
often  exhibited  by  the  bacillus  of  typhoid  fever  and  the 
bacillus  of  diphtheria.  After  inoculation  of  a  potato 
with  either  of  these  organisms  there  is  usually  no  naked- 
eye  evidence  of  growth,  though  microscopic  examina- 
tion of  scrapings  from  the  surface  of  the  potato  reveals 
an  active  multiplication  of  the  organisms  which  had 
been  planted  there.  The  potato  is  one  of  the  most  im- 
portant differential  media  that  we  possess  for  this  work. 


21 4  BA  CTERIOLOGY. 

CHANGES    IN    THE    REACTION    OF    MEDIA    AS    A    RESULT 
OF    BACTKRIAL    ACTIVITY. 

For  purposes  of  differentiation,  much  stress  is  laid 
upon  the  reaction  assumed  by  media  as  a  result  of  bac- 
terial growth.  Under  the  influence  of  certain  species 
the  medium  will  become  acid,  under  that  of  others  it  is 
alkaline,  while  some  cause  little  or  no  change.  In 
media  of  particular  composition — i.  e.,  those  containing 
traces  of  fermentable  carbohydrates,  notably  muscle- 
sugar,  as  seen  in  infusions  of  fresh  meat — the  reaction 
may  become  acid  with  the  beginning  of  growth  and 
subsequently  change  to  alkaline  after  the  supply  of 
fermentable  sugar  is  exhausted.  These  changes  of  reac- 
tion are  most  conveniently  observed  through  the  use  of 
indicators — bodies  that  either  lose  or  change  their  usual 
color  as  the  reaction  of  the  medium  to  which  they  are 
added  changes. 

Such  substances  as  litmus,  in  the  form  of  the  so-called 
"litmus  tincture,"  and  coralline  (rosolic  acid)  in  alco- 
holic solution,  are  commonly  employed  for  this  purpose. 
They  may  be  added  to  the  media  in  the  proportions 
given  in  the  chapter  on  Media,  and  the  changes  in  their 
colors  studied  with  different  bacteria.  Milk  and  litmus 
tincture  or  peptone  solution  to  which  rosolic  acid  has 
been  added  are  excellent  media  for  this  experiment. 

ANILINE  DYES  FOR  DIFFERENTIAL  DIAGNOSIS. — 
The  addition  to  solid  media  of  the  aniline  dyes,  fuchsin, 
methylene-blue,  methylene-green,  saffranin,  neutral  red, 
and  several  others,  as  well  as  combinations  of  these 
dyes,  has  been  recommended  as  a  means  of  differentia- 
tion of  bacteria.  The  changes  that  occur  as  a  result  of 
bacterial  growth  in  media  so  treated  consist  of  alterations 


FERMENT  A  TION.  21 5 

in  the  color  of  the  media,  due  to  the  oxidizing  or  reduc- 
ing properties  of  the  growing  bacteria.  It  is  doubtful 
if  this  is,  in  general,  an  important  diiferential  method ; 
at  all  events,  it  has  been  pretty  well  abandoned,  after 
having  enjoyed  at  one  time  some  degree  of  popularity, 
though  a  number  of  investigators  still  regard  saffranin 
and  neutral  red  as  useful  agents  for  the  differentiation 
of  allied  species,  and  as  handy  aids  in  the  identification 
of  those  species  capable  of  reducing  them. 

BEHAVIOR  TOWARD  STAINING-REAGENTS. — The  be- 
havior of  certain  bacteria  toward  the  different  dyes 
and  their  reactions  under  special  methods  of  after- 
treatment  aid  materially  in  their  identification.  With 
very  few  exceptions  bacteria  stain  readily  with  the  com- 
mon aniline  dyes ;  but  they  differ  markedly  in  the  te- 
nacity with  which  they  retain  these  colors  under  the 
subsequent  treatment  with  decolorizing-agents. 

The  tubercle  bacillus  and  the  bacillus  of  leprosy,  for 
example,  are  difficult  to  stain  ;  but  when  once  stained 
they  retain  their  color  under  the  action  of  such  energetic 
decolorizing-agents  as  alcohol,  nitric  acid,  oxalic  acid, 
etc.  Certain  other  organisms  when  stained  with  a  solu- 
tion of  gentian-violet  in  aniline-water  retain  their  color 
when  treated  with  such  decolorizing-bodies  as  iodine 
solution  and  alcohol  (Gram's  method),  while  again 
others  are  completely  decolorized  by  this  method. 
Many  of  them  can  only  be  washed  in  water,  or  but 
for  a  few  seconds  in  alcohol,  without  losing  their 
color.  It  is  essential  that  all  these  peculiarities  should 
be  carefully  noted  in  studying  an  organism. 

FERMENTATION. — The  production  of  gas  as  an  indi- 
cation of  fermentation  is  an  accompaniment  of  the 
growth  of  certain  bacteria.  This  is  best  studied  in 


21 6  BACTERIOLOGY. 

media  to  which  1  to  2  per  cent,  of  grape-sugar  (glucose) 
has  been  added.  A  convenient  method  of  demonstrat- 
ing this  property  is  to  employ  a  tube  about  half  full  of 
agar-agar  containing  the  necessary  amount  of  grape- 
sugar.  The  medium  is  to  be  liquefied  on  a  water-bath, 
and  then  cooled  to  about  42°  C.,  when  a  small  quantity 
of  a  pure  culture  of  the  organism  under  consideration 
should  carefully  be  distributed  through  it.  The  tube  is 
then  placed  in  ice-water  and  rapidly  solidified  in  the 
vertical  position.  When  solid  it  is  placed  in  the  incu- 
bator. After  twenty-four  to  thirty-six  hours,  if  the 
organism  possesses  the  property  of  causing  fermentation 
of  glucose,  the  medium  will  be  dotted  everywhere  with 
very  small  cavities  containing  the  gas  that  has  resulted. 
This  property  of  fermentation  with  evolution  of  gas 
is  of  such  importance  as  a  differential  characteristic 
that  considerable  attention  has  been  given  to  it,  and 
those  who  have  been  most  intimately  concerned  in  the 
development  of  our  knowledge  on  the  subject  do  not 
consider  it  sufficient  to  say  that  the  growth  of  an  organ- 
ism "is  accompanied  by  the  production  of  gas-bub- 
bles," but  that  under  given  conditions  we  should  deter- 
mine not  only  the  amount  of  gas  or  gases  produced 
by  the  organism  under  consideration,  but  also  their 
nature.  For  this  purpose,  Smith1  recommends  the 
employment  of  the  fermentation-tube.  This  is  a  tube 
bent  at  an  acute  angle,  closed  at  one  end  and  enlarged 
with  a  bulb  at  the  other.  At  the  bend  the  tube  is 
constricted.  To  it  a  glass  foot  is  attached  so  that 
the  tube  may  stand  upright,  (See  Fig.  37.)  To  fill 

1  An  excellent  and  exhaustive  contribution  to  this  subject  has  beer 
made  by  Theobald  Smith  in  the  Wilder  Quarter-Century  Book,  Ithaca, 
N.  Y.,  1893. 


FERMENTATION. 


217 


the  tube,  the  fluid  (it  is  used  only  with  fluid  media) 
is  poured  into  the  bulb  until  this  is  about  half  full. 
The  tube  is  then  tilted  until  the  closed  arm  is  nearly 
horizontal,  so  that  the  air  may  flow  out  into  the  bulb 
and  the  fluid  flow  into  the  closed  arm  to  take  its  place. 
When  this  has  been  completely  filled  sufficient  fluid 
should  be  added  to  bring  its  level  within  the  bulb  just 
beyond  the  bend,  and  the  opening  of  the  bulb  plugged 

FIG.  37. 


Fermentation-tube. 


with  cotton.  The  tubes  thus  filled  are  then  to  be  ster- 
ilized. During  sterilization  they  are  to  be  maintained 
in  the  upright  position.  Under  the  influence  of  heat  the 
tension  of  the  water-vapor  in  the  closed  arm  forces  most 
of  the  fluid  into  the  bulb.  As  the  tube  cools,  the  fluid 
returns  to  its  place  in  the  closed  arm  and  fills  it  again, 
with  the  exception  of  a  small  space  at  the  top,  which  is 
occupied  by  the  air  originally  dissolved  in  the  liquid 


218  BACTERIOLOGY. 

and  which  has  been  driven  out  by  the  heat.  The  air- 
bubble  should  be  tilted  out  after  each  sterilization  ;  and 
finally,  after  the  third  exposure  to  steam,  this  arm  of 
the  tube  will  be  free  from  air.  The  medium  employed 
is  bouillon  containing  some  fermentable  carbohydrate, 
as  glucose,  lactose,  or  saccharose.  After  inoculation  the 
flasks  are  placed  in  the  incubator,  and  the  amount  of 
gas  that  collects  in  the  closed  arm  is  noted  from  day  to 
day.  From  studies  that  have  been  made  this  gas  is 
found  to  consist  usually  of  about  one  part  by  volume  of 
carbonic  acid  and  two  parts  by  volume  of  an  explosive 
gas  consisting  largely  of  hydrogen.  For  determining  the 
nature  and  quantitative  relations  of  these  gases  Smith  * 
recommends  the  following  procedure  :  "  The  bulb  is 
completely  filled  with  a  2  per  cent,  solution  of  sodium 
hydroxide  (NaOH)  and  closed  tightly  with  the  thumb. 
The  fluid  is  shaken  thoroughly  with  the  gas  and  allowed 
to  flow  back  and  forth  from  bulb  to  closed  branch  and 
the  reverse  several  times,  to  insure  intimate  contact  of 
the  CO2  with  the  alkali.  Lastly,  before  removing  the 
thumb  all  the  gas  is  allowed  to  collect  in  the  closed  branch, 
so  that  none  may  escape  when  the  thumb  is  removed. 
If  CO2  be  present,  a  partial  vacuum  in  the  closed  branch 
causes  the  fluid  to  rise  suddenly  when  the  thumb  is  re- 
moved. After  allowing  the  layer  of  foam  to  subside 
somewhat  the  space  occupied  by  gas  is  again  measured, 
and  the  difference  between  this  amount  and  that  meas- 
ured before  shaking  with  the  sodium  hydroxide  solution 
gives  the  proportion  of  CO2  absorbed.  The  explosive 
character  of  the  residue  is  determined  as  follows :  the 
thumb  is  placed  again  over  the  mouth  of  the  bulb  and 
the  gas  from  the  closed  branch  is  allowed  to  flow  into 

1  Loc.  cit,  p.  190. 


FERMENT  A  TION. 


219 


FIG.  38. 


the  bulb  and  mix  with  the  air  there  present.     The  plug 
is  then  removed  and  a  lighted  match  inserted  into   the 
mouth   of  the    bulb.     The  intensity  of 
the  explosion  varies  with  the  amount  of 
air  present  in  the  bulb." 

Durham's  Fermentation-tube.  — Dur- 
ham employs  a  convenient  modification 
of  the  ordinary  fermentation-tube,  which 
is  constructed  in  the  following  man- 
ner :  test-tubes  of  about  10  or  12  c.c. 
capacity  are  placed  in  an  inverted  posi- 
tion within  a  larger  test-tube,  and  the 
latter  plugged  with  cotton  in  the  usual 
way  and  sterilized.  (See  Fig.  38.)  The 
small  tube  should  fit  loosely  within  the 
larger  one.  The  medium  to  be  used  is 
run  into  the  larger  tube  until  there  is 
present  about  50  per  cent,  more  than  the 
volume  of  the  smaller  tube.  The  whole 
is  then  sterilized  in  streaming  steam  by 
the  fractional  method.  After  the  first 
sterilization  the  small  tube  will  be  found 
almost  filled  with  fluid,  over  which  a 
small  air-bubble  lies.  After  the  second 
or  third  sterilization  this  air-bubble  is 
completely  expelled,  and  the  small  tube 
contains  nothing  but  the  liquid. 

The   medium   that   Durham    employs 
for  the  fermentation-test  is  a  1  per  cent, 
solution  of  Witte's  peptone  in   distilled 
water,  to  which   have  been   added  known  amounts  of 
some   such   fermentable   sugar   as   glucose,    saccharose, 
lactose,   mannite,   etc.,  as  the  case  may  demand.     He 


220  BACTERIOLOGY. 

prefers  peptone  to  meat-infusion  bouillon  for  the  reason 
that  the  latter  often  contains  traces  of  muscle-sugar, 
and  thereby  is  likely  to  complicate  the  results.  He 
prefers  neutralization  with  organic  acids  rather  than 
mineral  acids,  and  uses  citric  acid  by  preference,  the 
reason  for  this  being  that  where  sugars  such  as  those 
mentioned  are  acted  upon  by  mineral  acids  under  the 
influence  of  heat  their  composition  is  apt  to  be  altered. 

NOTE. — Prepare  two  fermentation-tubes  as  follows : 
Fill  one  with  a  1  per  cent,  watery  solution  of  peptone 
to  which  2  per  cent,  of  glucose  has  been  added ;  fill 
the  other  with  a  similar  peptone  solution,  but  to  which 
only  0.3  per  cent,  of  glucose  has  been  added.  Sterilize 
and  inoculate  with  bacillus  coli  communis.  How  do 
the  two  tubes  diifer  from  one  another  after  eighteen  to 
twenty-four  hours  in  the  incubator?  First,  as  regards 
the  reaction  of  the  fluid  in  the  open  arms  of  the  tubes. 
Second,  as  to  accumulation  of  gas  in  closed  arms  of  the 
tubes.  Third,  as  to  the  capacity  of  each  solution  for 
reducing  copper  in  Fehling's  solution.  What  differ- 
ences are  observed,  and  how  may  they  be  explained? 

CULTIVATION  WITHOUT  OXYGEN. — As  we  have 
already  learned,  there  is  a  group  of  bacteria  to  which 
the  designation  "anaerobic"  has  been  given,  which 
are  characterized  by  inability  to  grow  in  the  presence 
of  free  oxygen.  For  the  cultivation  of  the  members  of 
this  group,  a  number  of  devices  are  employed  for  the 
exclusion  of  free  oxygen  from  the  cultures. 

Koch's  method.  Koch  covered  the  surface  of  a  gela- 
tin plate,  which  had  been  previously  inoculated,  with 
a  thin  sheet  of  sterilized  isinglass.  The  organisms 


CULTIVATION   WITHOUT  OXYGEN.  221 

which  grew  beneath  it  were  supposed  to  develop  with- 
out oxygen. 

Hesse's  method.  Hesse  poured  sterilized  oil  upon  the 
surface  of  a  culture  made  by  stabbing  a  tube  of  gelatin. 
The  growth  that  occurred  along  the  track  of  the  needle 
was  supposed  to  be  anaerobic  in  nature. 

FIG.  39. 


Liborius  tube  for  anaerobic  cultures. 

Methods  of  Liborius.  Liborius  has  suggested  two 
useful  methods  for  this  purpose.  One  is  to  nearly  (about 
three-quarters)  fill  a  test-tube  with  gelatin  or  agar-agar, 
which,  after  having  been  sterilized,  is  to  be  kept  in  a 
vessel  of  boiling  water  for  ten  minutes  to  expel  all  air 
from  it.  It  is  then  rapidly  cooled  in  ice- water,  and 
when  between  30°  and  40°  C.,  still  fluid,  is  to  be  inoc- 
ulated and  very  rapidly  solidified.  It  is  then  sealed  in 
the  flame.  Anaerobic  bacteria  develop  only  in  the  lower 


222  BACTERIOLOGY. 

layers  of  the  medium.  In  his  other  method  he  employs 
a  special  tube,  known  as  "  the  Liborius  tube."  Its  con- 
struction is  shown  in  Fig.  39. 

Through  the  side  tube  hydrogen  is  passed  until  it  re- 
places all  the  air  ;  the  contracted  parts,  both  of  the  neck 
of  the  tube  and  the  side  arm,  are  then  sealed  in  the 
flame.1  This  tube  can  be  used  for  either  solid  or  liquid 
media,  but,  owing  to  its  usual  small  capacity,  gives 
better  results  with  fluid  media.  (For  precautions  in 
using  hydrogen,  see  note  to  FrankePs  method,  page 
223.) 

Method  of  Buchner.  The  plan  suggested  by  Buchner, 
of  allowing  the  cultures  to  develop  in  an  atmosphere 
robbed  of  its  oxygen  by  pyrogallic  acid,  gives  very  good 
results.  In  this  method  the  culture,  which  is  either  a 
slant-  or  stab-culture  in  a  test-tube,  is  placed — tube, 
cotton  plug,  and  all — into  a  larger  tube,  in  the  bottom 
of  which  have  been  deposited  1  gramme  of  pyrogallic 
acid  and  10  c.c.  of  -fa  normal 2  caustic-potash  solution. 

1  As  the  tubes  come  from  the  maker  the  contracted  parts  marked  x 
in  the  cut  are  usually  so  thick  as  to  render  the  sealing  in  the  flame 
during  the  passage  of  hydrogen  somewhat  troublesome ;  it  is  better 
to  draw  them  out  in  the  flame  quite  thin  before  passing  the  hydrogen 
into  the  tube.     This  makes  the  final  sealing  a  matter  of  no  difficulty. 

2  A  normal  solution  is  one  that  contains  in  a  litre  as  many  grammes 
of  the  dissolved  substance  as  are  indicated  by  its  molecular  equivalent. 
The  equivalent  is  that  amount  of  a  chemical  compound  which  possesses 
the  same  chemical  value  as  does  one  atom  of  hydrogen.    For  example  : 
one  molecule  of  hydrochloric  acid  (HC1)  has  a  molecular  weight  and 
also  an  equivalent  weight  of  36.5 ;  a  molecule  of  this  acid  has  the 
same  chemical  value  as  one  atom  of  hydrogen.     Its  normal  solution  is 
therefore  36.5  grammes  to  the  litre.    On  the  other  hand,  sulphuric  acid 
(HzSOi)  contains  in  each  molecule  two  replaceable  hydrogen  atoms ; 
its  normal  solution  is  not,  therefore,  80  grammes  (its  molecular  weight) 
to  the  litre,  but  that  amount  which  would  be  equivalent  chemically  to 
one  hydrogen  atom,  viz.,  40  grammes  (one-half  its  molecular  weight) 
to  the  litre.     A  normal  solution  of  caustic  potash  contains  as  many 


CULTIVATION   WITHOUT  OXYGEN. 


223 


The  larger  tube  is  then  tightly  plugged  with  a  rubber 
stopper.  The  oxygen  is  quickly  absorbed  by  the  pyro- 
gallic  acid,  and  the  organisms  develop  in  the  remaining 
constituents  of  the  atmosphere,  viz.,  nitrogen,  a  small 
amount  of  CO2,  and  a  trace  of  ammonia. 

FIG.  40. 


Frankel's  method  for  the  cultivation  of  anaerobic  bacteria. 

^[ethod  of  C.  Frdnkel.  Carl  Frankel  suggests  the 
following  as  a  modification  of  or  substitute  for  the  tube 
of  Liborius :  the  tube  is  first  inoculated  as  if  it  were 
to  be  poured  as  a  plate  or  rolled  as  an  ordinary  Esmarch 
tube.  The  cotton  plug  is  then  replaced  by  a  rubber 
stopper,  through  which  pass  two  glass  tubes.  These 
must  all  have  been  sterilized  in  the  steam  sterilizer 

grammes  to  the  litre  as  the  number  of  its  molecular  weight — 56.1 
grammes  to  the  litre  of  water. 


224  BACTERIOLOGY. 

before  using.  On  the  outer  side  of  the  stopper  these 
two  tubes  are  bent  at  right  angles  to  the  long  axis  of 
the  test-tube  into  which  they  are  to  be  placed,  and  both 
are  slightly  drawn  out  in  a  gas-flame.  Both  of  these 
tubes  must  be  provided,  before  sterilization,  with  a 
plug  of  cotton  ;  this  is  to  prevent  the  access  of  foreign 
organisms  to  the  medium  during  manipulations.  At 
the  inner  side  of  the  rubber  stopper — that  is,  the  end 
which  is  to  be  inserted  into  the  test-tube — the  glass 
tubes  are  of  different  lengths  :  one  reaches  to  within 
0.5  cm.  of  the  bottom  of  the  test-tube,  the  other  is  cut 
off  flush  with  the  under  surface  of  the  stopper.  The 
outer  end  of  the  longer  glass  tube  is  then  connected 
with  a  hydrogen  generator  and  hydrogen  is  allowed 
to  bubble  through  the  gelatin  (Fig.  40,  A)  in  the  tube 
until  all  contained  air  has  been  expelled  and  its  place 
taken  by  the  hydrogen.1  When  the  hydrogen  has  been 

1  Before  beginning  the  experiment  it  is  always  wise  to  test  the  hydro- 
gen— i.  e.,  to  see  that  it  is  free  from  oxygen  and  that  there  is  no  danger 
of  an  explosion,  for  unless  this  be  done  the  entire  apparatus  may  be 
blown  to  pieces  and  a  serious  accident  occur.  The  agents  used  should 
be  pure  zinc  and  pure  sulphuric  acid  of  about  25  to  30  per  cent, 
strength.  With  the  primary  evolution  of  the  gas  the  outlet  of  the 
generator  should  be  closed  and  kept  closed  until  the  gas  reservoir  is 
quite  filled  with  hydrogen.  The  outlet  should  then  be  opened  and  the 
entire  volume  of  gas  allowed  to  escape,  care  being  taken  that  no  flame 
is  in  the  neighborhood.  This  should  be  repeated,  after  which  a  sample 
of  the  hydrogen  generated  should  be  collected  in  an  inverted  test-tube 
in  the  ordinary  way  for  collecting  gases  over  water,  viz.,  by  filling  a 
test-tube  with  water,  closing  its  mouth  with  the  thumb,  inverting  it, 
and  placing  its  mouth  under  water,  when,  after  removing  the  thumb, 
the  water  will  be  kept  in  it  by  atmospheric  pressure.  The  hydrogen 
which  is  flowing  from  the  open  generator  may  be  conducted  to  the  test- 
tube  by  rubber  tubing.  When  the  water  has  been  replaced  test  the  gas 
by  holding  a  flame  near  the  open  mouth  of  the  test-tube.  If  no  explo- 
sion occurs,  the  hydrogen  is  safe  to  use.  Should  there  be  an  explosion. 
the  generation  of  hydrogen  must  be  continued  in  the  apparatus  until 
it  burns  with  a  colorless  flame  when  tested  in  a  test-tube. 


CULTIVATION  WITHOUT  OXYGEN.  225 

bubbling  through  the  gelatin  for  about  five  minutes 
(at  least)  one  can  be  reasonably  sure  that  all  oxygen 
has  been  expelled.  The  drawn-out  portions  of  the 
tubes  can  then  be  sealed  in  the  gas-flame  without  fear 
of  an  explosion.  The  protruding  end  of  the  rubber 
stopper  is  then  painted  around  with  melted  paraffin 
and  the  tube  rolled  in  the  way  given  for  ordinary 
Esmarch  tubes.  A  tube  thus  prepared  and  containing 
growing  colonies  is  shown  in  Fig.  40,  B. 

The  development  that  now  occurs  is  in  an  atmos- 
phere of  hydrogen,  all  oxygen  having  been  expelled. 
During  the  operation  the  tube  containing  the  liquefied 
gelatin  should  be  kept  in  a  water-bath  at  a  temperature 
sufficiently  high  to  prevent  its  solidifying,  and  at  the 
same  time  not  high  enough  to  kill  thf;  organisms  with 
which  it  has  been  inoculated. 

One  of  the  obstacles  to  the  successful  performance  of 
this  method  is  the  bubbling  of  the  gelatin,  the  foam 
from  which  will  often  fill  the  exit-tube  and  sometimes 
be  forced  from  it.  This  may  be  obviated  by  reversing 
the  order  of  proceeding,  viz.  :  roll  the  Esmarch  tube 
in  the  ordinary  way  with  the  organisms  to  be  studied, 
using  a  relatively  small  amount  of  gelatin,  so  as  to 
have  as  thin  a  layer  as  possible  when  it  is  rolled. 
Then  replace  the  cotton  plug  with  the  sterilized  rubber 
stopper  carrying  the  glass  tubes  through  which  the 
hydrogen  is  to  be  passed,  and  allow  the  hydrogen  to 
flow  through  as  in  the  method  first  given.  The  gas 
now  passes  over  the  gelatin  instead  of  through  it, 
and  consequently  no  bubbling  results.  In  all  other 
respects  the  procedure  is  the  same  as  that  given  by 
Frankel. 

Method   of  Kitasato   and    Weil.     For   favoring   an- 

15 


226  BACTERIOLOGY. 

aerobic  conditions  Kitasato  and  Weil  have  suggested 
the  addition  to  the  culture-media  of  some  strong  re- 
ducing-agent.  They  recommend  formic  acid  or  sodium 
formate  in  0.3  to  0.5  per  cent. ;  glucose  in  1.5  to  2  per 
cent. ;  or  blue  litmus  tincture  in  5  per  cent,  by  volume. 
This  is,  of  course,  in  addition  to  an  atmosphere  from 
which  all  oxygen  has  been  expelled.  As  a  reducing- 
agent  for  this  purpose,  Theobald  Smith  regards  a  weaker 
solution  of  glucose,  0.3  to  0.5  per  cent.,  as  more  ad- 
vantageous ;  and  Wright  obtains  better  results  when 
glucose  is  added  if  the  primary  reaction  of  the  media 
is  about  neutral  to  phenolphtalein. 

Esmarch's  method.  Esmarch's  plan  is  to  prepare  in 
the  usual  way  a  roll-tube  of  the  organisms ;  subject  it 
to  a  low  temperature,  and  while  quite  cold  fill  it  with 
liquefied  gelatin,  which  is  caused  to  solidify  rapidly.  In 
this  method  the  colonies  develop  along  the  sides  of  the 
tubes,  and  can  more  easily  be  studied  than  when  they  are 
scattered  through  the  gelatin,  as  in  the  method  of  Liborius. 

Method  of  Park.  A  very  simple,  convenient,  and 
efficient  method  is  employed  by  Park.  It  consists  in 
covering  the  medium  in  which  the  anaerobic  species  are 
to  be  cultivated  with  liquid  paraffin  (albolene).  The 
best  results  are  obtained  when  the  amount  of*  paraffin 
added  is  about  half  that  of  the  liquid  in  the  tube  or 
flask.  The  liquid  paraffin  has  the  advantage  over  the 
solid  paraffin  in  not  retracting  from  the  walls  of  the 
vessel  on  cooling.  All  air  is  expelled  from  flasks  or 
tubes  prepared  in  this  way,  by  heating  them  in  the  auto- 
clave. The  layer  of  paraffin  prevents  the  reabsorption 
of  oxygen  driven  off  by  the  heat.  After  cooling,  the 
inoculation  is  made  by  passing  the  needle  through  the 
paraffin  well  down  into  the  media. 


INDOL  PRODUCTION.  227 

By  some  workers  the  oxygen  is  removed  from  the 
culture-medium  by  the  use  of  the  air-pump. 

Many  other  methods  are  employed  for  this  special 
purpose,  but  for  the  beginner  those  given  will  suffice. 

From  what  has  been  said,  it  may  be  inferred  that  the 
cultivation  of  anaerobic  bacteria  is  a  simple  matter 
attended  with  but  little  difficulty.  Such  an  opinion 
will,  however,  be  quickly  abandoned  when  the  beginner 
attempts  this  part  of  his  work  for  the  first  time,  and 
particularly  when  his  efforts  are  directed  toward  the 
separation  of  these  forms  from  other  organisms  with 
which  they  are  associated.  The  presence  of  spore- 
forming,  facultative  anaerobes  in  mixed  cultures  is 
always  to  be  suspected,  and  it  is  this  group  that  renders 
the  task  so  difficult.  At  best  the  work  requires  undi- 
vided attention  and  no  small  degree  of  skill  in  bacterio- 
logical technique. 

INDOL  PRODUCTION. — The  generation  of  products 
other  than  those  that  give  rise  to  alterations  in  the  reac- 
tion of  the  media,  and  whose  presence  may  be  detected 
by  chemical  reactions,  is  now  a  recognized  step  in  the 
identification  of  different  species  of  bacteria.  Among 
these  products  is  one  that  is  produced  by  a  number 
of  organisms,  and  whose  presence  may  easily  be  de- 
tected by  its  characteristic  behavior  when  treated  with 
certain  substances.  I  refer  to  nitroso-indol,  the  reac- 
tions of  which  were  described  by  Beyer  in  1869,  and 
the  presence  of  which  as  a  product  of  the  growth  of 
certain  bacteria  has  since  furnished  a  topic  for  consid- 
erable discussion. 

Indol,  the  name  by  which  this  body  is  now  generally 
known,  when  acted  upon  by  reducing-agents  becomes 
of  a  more  or  less  decided  rose  color.  This  body  was 


228  B  A  CTERLOLOG  Y. 

recognized  as  one  of  the  products  of  growth  of  the 
spirillum  of  Asiatic  cholera  first  by  Poel,  and  a  short 
time  subsequently  by  Bujwid  and  by  Dunham,  and  for 
a  time  was  believed  to  be  peculiarly  characteristic  of 
the  growth  of  this  organism.  It  has  since  been  found 
that  there  are  many  other  bacteria  which  also  possess 
the  property  of  producing  indol  in  the  course  of  their 
development.  It  is  constantly  present  in  putrefying 
matters,  and  is  one  of  the  aromatic  bodies  that  give  to 
faeces  their  characteristic  odor. 

The  methods  employed  for  its  detection  are  as  follows  : 
cultivate  the  organism  for  twenty-four  to  forty-eight 
hours  at  a  temperature  of  37°  C.,  in  the  simple  pep- 
tone solution  known  as  "  Dunham's  solution "  (see 
formula  for  this  medium).  This  solution  is  preferred 
because  its  pale  color  does  not  mask  the  rose  color  of 
the  reaction  when  the  amount  of  indol  present  is  very 
small. 

Four  tubes  should  always  be  inoculated  and  kept 
under  exactly  the  same  conditions  for  the  same  length 
of  time. 

At  the  end  of  twenty-four  or  forty-eight  hours  the 
test  may  be  made.  Proceed  as  follows  :  to  a  tube  con- 
taining 7  c.c.  of  the  peptone  solution,  but  which  has  not 
been  inoculated,  add  10  drops  of  concentrated  sulphuric 
acid.  To  another  similar  tube  add  1  c.c.  of  a  0.01  per 
cent,  solution  of  sodium  nitrite,  and  afterward  10  drops 
of  concentrated  sulphuric  acid.  Observe  the  tubes  for 
five  to  ten  minutes.  No  alteration  in  their  color  ap- 
pears, or  at  least  there  is  no  production  of  a  rose  color. 
They  contain  no  indol. 

Treat  in  the  same  way,  with  the  acid  alone,  two  of 
the  tubes  which  have  been  inoculated.  If  no  rose  color 


INDOL  PRODUCTION.  229 

appears  after  five  or  ten  minutes,  add  1  c.c.  of  the 
sodium  nitrite  solution.  If  now  no  rose  color  is  pro- 
duced, the  indol  reaction  may  be  considered  as  negative 
— i.  e.,  no  indol  has  been  formed  as  a  product  of  the 
growth  of  the  bacteria. 

If  indol  is  present,  and  the  rose  color  appears  after 
the  addition  of  the  acid  alone,  it  is  plain  that  not  only 
indol  has  been  formed,  but  coincidently  a  reducing- 
body.  This  is  found,  by  proper  means,  to  be  nitrous 
acid.  The  sulphuric  acid  liberates  this  acid  from  its 
salts  and  permits  of  its  reducing  action  being  brought 
into  play. 

If  the  rose  color  appears  only  after  the  addition  of 
both  the  acid  and  the  nitrite  solution,  then  indol  has 
been  formed  during  the  growth  of  the  organisms,  but 
no  nitrites. 

Control  the  results  obtained  by  treating  the  two 
remaining  cultures  in  the  same  way. 

The  test  is  sometimes  made  by  allowing  concentrated 
sulphuric  acid  to  flow  down  the  sides  and  collect  at  the 
bottom  of  the  tube ;  the  reaction  is  then  seen  as  a  rose- 
colored  zone  overlying  the  line  of  contact  of  the  acid  and 
culture-medium.  This  method  is  open  to  the  objection 
that,  if  indol  is  present  in  only  a  very  small  amount, 
the  faint  rose  tint  produced  by  it  is  apt  to  be  masked 
by  a  brown  color  that  results  from  the  charring  action 
of  the  concentrated  acid  on  the  other  organic  matters 
in  the  culture-medium,  so  that  its  presence  may  in  this 
way  escape  detection.  In  view  of  this,  Petri  recom- 
mends the  use  of  dilute  sulphuric  acid.  He  states  that 
when  indol  is  present  the  characteristic  rose  color  ap- 
pears a  little  more  slowly  \vfth  the  dilute  acid,  but 
it  is  more  permanent,  and  there  is  never  any  like- 


230  BA  CTERIOL  0  G  Y. 

Jihood  of  its  presence  being  masked  by  other  color- 
reactions. 

Muir  and  Ritchie  recommend  the  use  of  ordinary 
fuming  or  yellow  nitric  acid  for  this  test.  In  this 
method  two  or  three  drops  of  the  acid  are  added  to 
the  culture  under  consideration.  If  indol  be  present, 
the  red  color  appears  as  a  result  of  the  reducing  action 
of  the  nitrous  acid  upon  it.  The  defect  in  this  method 
is  that  it  reveals  only  the  presence  of  indol,  and  fails  to 
indicate  whether  or  not  reducing-bodies  were  coinci- 
dently  formed  with  the  indol.  As  a  test  for  indol  alone 
it  is  convenient  and  entirely  trustworthy. 

REDUCING  POWER  OF  BACTERIA. — The  power  to 
reduce  chemical  compounds  from  a  higher  to  a  lower 
state  may  be  said  to  be  common  to  all  bacteria.  In 
some  bacteria,  perhaps  the  majority,  it  is  most  conspicu- 
ously manifested  in  connection  with  substances  contain- 
ing sulphur,  hydrogen  sulphide  being  formed.  In  other 
bacteria  it  is  best  seen  in  connection  with  the  alterations 
produced  in  certain  pigments,  as  litmus,  methylene- 
blue,  indigo,  etc.,  the  normal  color  disappearing  in  part 
or  entirely  according  to  the  nature  and  activity  of  the 
process.  Other  bacteria  have  the  property  of  reducing 
certain  salts,  as  in  the  reduction  of  nitrates  to  nitrites, 
or  even  to  ammonia  by  the  denitrifying  bacteria.  In 
some  instances  these  reductions  result  from  the  fact  that 
the  bacteria  liberate  hydrogen  from  the  compounds,  in 
others  it  results  from  the  fact  that  the  bacteria  abstract 
oxygen  from  such  compounds,  while  in  still  other  instances 
the  reduction  is  of  a  more  complex  nature.  Each  of  these 
changes,  therefore,  indicates  the  nature  of  some  of  the  me- 
tabolic activities  manifested  by  the  bacteria  in  question. 

Some  of  these  reductions  may   be   detected   by  the 


REDUCING  POWER   OF  BACTERIA.  231 

application  of  comparatively  simple  tests  for  the  pres- 
ence of  the  end-products. 

Test  with  Pigments. — The  reduction  of  various  pig- 
ments and  aniline  dyes  is  usually  manifested  by  altera- 
tions in  the  depth  of  color  or  in  the  complete  decoloriza- 
tion  of  the  pigment.  In  some  instances  the  reduction 
is  first  manifested  in  the  depth  of  the  medium,  and  in 
such  instances  the  natural  color  of  the  pigment  may 
frequently  be  restored  on  shaking  the  medium.  This 
is  manifestly  a  deoxidation  of  the  pigment  arising  from 
the  avidity  of  the  bacteria  for  oxygen  when  growing  in 
the  depth  of  the  medium.  In  other  instances  the  reduc- 
tion is  more  complete,  and  simple  agitation  of  the 
medium  fails  to  restore  the  original  color. 

Test  for  Hydrogen  Sulphide. — The  reduction  of  sul- 
phur compounds  may  be  determined  by  growing  the 
bacteria  in  peptone  solution  containing  ferric  tartrate, 
when  the  presence  of  hydrogen  sulphide  will  be  indi- 
cated by  the  brownish-black  or  jet-black  color  of  the 
precipitated  iron-sulphide. 

The  complete  reduction  of  nitrates  is  brought  about 
by  many  bacteria.  Other  bacteria  are  capable  of  carry- 
ing the  reducing  action  as  far  as  the  formation  of  ammo- 
nia, while  still  others  merely  reduce  the  nitrates  to 
nitrites.  These  reducing  functions  are  encouraged  and 
may  be  demonstrated  by  cultivating  the  bacteria  in  pep- 
tone solution  containing  potassium  nitrate. 

Test  for  Nitrites. — The  method  of  Griess,as  modified  by 
1 1<  >svay,  is  quite  satisfactory.  These  reagents  are  required : 

( a)  Naphthylamine,  0.1  gramme. 
Distilled  water,                                 20.0  c.c. 
Acetic  acid  (25  per  cent,  solution),  150.0   " 

(b)  Sulfanilic  acid,  0.5  gramme. 
Acetic  acid  (25  per  cent,  solution),  150.0  c.c. 


232  BACTERIOLOGY. 

In  preparing  solution  a  the  naphthylamine  is  dis- 
solved in  20  c.c.  of  boiling  water,  filtered,  allowed  to 
cool,  and  mixed  with  the  dilute  acetic  acid.  Solutions 
a  and  b  are  then  mixed.  It  is  best  prepared  as  needed, 
though  it  may  be  preserved  for  some  time  in  a  glass- 
stoppered  bottle. 

In  testing  for  nitrites  the  reagent  is  added  in  the 
proportion  of  one  volume  of  reagent  to  five  volumes 
of  culture.  When  nitrites  have  been  formed  a  deep- 
red  color  appears  in  a  few  seconds.  If  no  nitrites  have 
been  formed  the  culture  remains  colorless.  In  testing 
cultures  it  is  always  necessary  to  control  the  results  by 
blank  tests  on  a  portion  of  the  same  medium  that  had 
not  been  inoculated,  as  some  of  the  ingredients  of  the 
medium  may  have  contained  nitrites. 

Another  test  for  the  formation  of  nitrites  is  a  mixt- 
ure of  starch  and  potassium  iodide,  as  follows  : 

Starch,  2.0  grammes. 

Potassium  iodide,  0.5         " 

Water,  100.0  c.c. 

Warm  the  mixture  until  the  starch  is  completely  dis- 
solved. 

In  testing  for  nitrites  add  0.5  c.c.  of  the  reagent  to  a 
tube  of  culture,  and  follow  this  by  the  addition  of  2  or 
3  drops  of  pure  sulphuric  acid.  If  nitrites  have  been 
formed,  a  dark-blue  or  purple  color  will  appear.  Con- 
trol-tubes of  the  medium  show  no  color  reaction,  or 
merely  a  trace  of  blue  coloration. 

Test  for  Ammonia. — The  formation  of  ammonia  may 
be  detected  by  testing  with  Nessler's  reagent.  The 
most  satisfactory  results  are  obtained  by  cultivating 


REDUCING   POWER   OF  BACTERIA.  233 

the  organisms  in  a  litre  of  culture  fluid  and  then 
distilling  off  portions  of  the  culture,  collecting  in 
Nessler  tubes,  and  applying  1  c.c.  of  the  reagent  to 
each  50  c.c.  of  the  distillate.  The  presence  of  ammo- 
nia in  the  distillate  is  shown  by  the  yellow  coloration 
resulting  from  the  addition  of  the  reagent. 

The  direct  application  of  the  reagent  to  the  culture 
will  give  satisfactory  results  if  a  great  deal  of  ammonia 
lias  been  formed.  In  this  instance  the  mercury  in 
the  reagent  will  be  precipitated  as  mercurous  oxide. 
Another  rough  test  for  the  formation  of  ammonia  is  to 
place  a  strip  of  filter-paper — moistened  with  the  Ness- 
ler reagent — over  the  mouth  of  a  test-tube  containing 
the  culture,  and  then  gently  heating  the  culture.  As 
the  ammonia  is  driven  off  by  the  heat,  it  will  react  on 
the  reagent  on  the  strip  of  paper. 

EXAMINATION  OF  CULTURES  FOR  BACTERIAL  TOX- 
INS.— In  the  systematic  study  of  a  pathogenic  organism 
it  is  necessary  to  know  whether  it  is  capable  of  pro- 
ducing a  soluble  toxin  when  growing  in  culture-media. 
This  is  done  by  filtering  cultures  of  various  ages  and 
testing  the  effect  of  the  filtrate  upon  susceptible 
animals. 

FILTRATION  OF  CULTURES. — A  variety  of  filters 
have  been  devised  for  the  purpose  of  filtering  liquid 
cultures  and  other  fluids  to  obtain  sterile  filtrates. 
These  filters  are  usually  constructed  of  unglazed  porce- 
lain or  of  infusorial  earth,  and  are  made  in  the  form  of 
hollow  cylinders  or  bulbs.  The  best-known  forms  of 
bacterial  filters  are  those  of  Chamberland  and  of  Berke- 
feld.  All  the  filters  used  for  this  purpose  require  some 
motive  power  to  force  the  fluid  through  the  filter.  Com- 
prossed  air  may  be  employed  to  force  the  fluid  through 


234  BACTERIOLOGY. 

the  filter,  or  atmospheric  pressure  may  be  utilized  by 
creating  a  negative  pressure  on  the  distal  side  of  the 
filter  by  the  use  of  an  air-pump. 

It  is  always  necessary  to  test  the  sterility  of  the  fil- 
trate by  making  cultures  from  it  into  nutritive  media 
and  noting  whether  growth  takes  place  or  not, 


CHAPTEK   XII. 

Inoculation  of  animals — Subcutaneous  inoculation — Intravenous  in- 
jection— Inoculation  into  the  great  serous  cavities,  and  into  the 
anterior  chamber  of  the  eye — Observation  of  animals  after  inocu- 
lation. 

AFTER  subjecting  an  organism  to  the  methods  of 
study  that  we  have  thus  far  reviewed  there  remains  to 
be  tested  its  action  upon  animals — i.  e.,  to  determine  if 
it  possesses  the  property  of  producing  disease  or  not ; 
and,  if  so,  what  are  the  pathological  results  of  its 
growth  in  the  tissues  of  these  animals,  and  in  what  way 
must  it  gain  entrance  to  the  tissues  in  order  to  produce 
these  results  ?  The  mode  of  deciding  these  points  is  by 
inoculation,  which  is  practised  in  different  ways  accord- 
ing to  circumstances.  Most  commonly  a  bit  of  the  cult- 
ure to  be  tested  is  simply  introduced  beneath  the  skin 
of  the  animal ;  but  in  other  cases  it  may  be  necessary 
to  introduce  it  directly  into  the  vascular  or  lymphatic 
circulation,  or  into  one  or  the  other  of  the  great  serous 
cavities ;  or,  for  still  other  purposes  of  observation,  into 
the  anterior  chamber  of  the  eye,  upon  the  iris  or  within 
the  skull  cavity,  upon  .the  dura  or  brain  substance. 

SUBCUTANEOUS  INOCULATION  OF  ANIMALS. — The 
animals  usually  employed  in  the  laboratory  for  purposes 
of  inoculation  are  white  mice,  gray  house-mice,  guinea- 
pigs,  rabbits,  and  pigeons. 

For  simple  subcutaneous  inoculation  the  steps  in  the 
process  are  practically  the  same  in  all  cases.  The  hair  or 
feathers  are  to  be  carefully  removed.  If  the  skin  is  very 

235 


236  BA  CTERIOLOG  Y. 

dirty,  it  may  be  scrubbed  with  soap  and  water.  Steriliza- 
tion of  the  skin  is  practically  impossible,  so  it  need  not 
be  attempted.  If  the  inoculation  is  to  be  made  by  means 
of  a  hypodermic  syringe,  then  a  fold  of  the  skin  may  be 
lifted  up  and  the  needle  inserted  in  the  usual  way.  If  a 
solid  culture  is  to  be  inoculated,  a  fold  of  skin  may  be 
taken  up  with  forceps  and  a  pocket  cut  into  it  with 
scissors  which  have  previously  been  sterilized.  This 
pocket  must  be  cut  large  enough  to  admit  the  end  of  the 
needle  without  its  touching  the  sides  of  the  opening  as  it 
is  inserted.  Beneath  the  skin  will  be  found  the  super- 
ficial and  deep  connective-tissue  fascia.  These  must  be 
taken  up  with  sterilized  forceps,  and  with  sterilized  scis- 
sors incised  in  a  way  corresponding  to  the  opening  in  the 
skin.  The  pocket  is  then  to  be  held  open  with  the  for- 
ceps and  the  substance  to  be  inserted  is  introduced  as 
far  under  the  skin  and  fasciae  as  possible,  care  being 
taken  not  to  touch  the  edges  of  the  wound  if  it  can 
be  avoided.  The  edges  of  the  wound  may  then  be 
simply  pulled  together  and  allowed  to  remain.  No 
stitching  or  efforts  at  closing  it  are  necessary,  though  a 
drop  of  collodion  over  the  point  of  operation  may  serve 
to  lessen  contamination. 

During  manipulation  the  animal  must  be  held  still. 
For  this  purpose  special  forms  of  holders  have  been 
devised ;  but  if  an  assistant  is  at  hand,  the  simple  sub- 
cutaneous inoculation  may  be  made  without  the  aid  of 
a  mechanical  holder. 

It  is  at  times,  however,  more  convenient  to  dispense 
with  an  assistant ;  one  of  several  forms  of  apparatus  that 
have  been  devised  for  holding  mice,  guinea-pigs,  rats, 
rabbits,  etc.,  may  then  be  used.  For  small  animals,  such 
as  mice  and  rats,  the  holder  suggested  by  Kitasato  is  very 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS.  287 

useful .    It  is  simply  a  metal  plate  attached  to  a  stand  by  a 
clamped  ball-and-socket  joint,  so  that  it  can  be  fixed  in 


FIG.  41. 


Kitusato's  mouse-holder. 


any  position.     It  is  provided  with  a  spring-clip  at  one 
end  that  holds  the  animal  by  the  skin  of  the  neck,  and 


FIG.  42. 


Holder  for  larger  animals. 

at  the  other  end  with  another  clamp  that  holds  the  tail 
of  the  animal.     This  holder  is  shown  in  Fig.  41.     For 


238 


BACTERIOLOGY. 


larger  animals  the  form  of  holder  shown  in  Fig.  42  is 
commonly  used. 

The  holder  devised  by  Sweet,1  which  can  be  made  of 
any  size,  from  that  suitable  to  a  guinea-pig  up  to  that 
large  enough  to  secure  a  dog,  is  in  every  way  the  most 
convenient  that  we  have  encountered  and,  from  the 
standpoint  of  the  animal,  is  the  most  humane.  It  con- 


FIG.  43. 


sists  of  four  pieces  of  heavy  round  wire  so  bent  that  two 
engage  the  animal  immediately  behind  the  lower  jaw 
while  the  remaining  two  close  over  the  muzzle.  All 
are  held  in  position  by  a  single  clamp  controlled  by  a 
single  thumb-screw.  When  the  screw  is  reversed  and 

1  Sweet :  "  A  simple,  humane  holder  for  small  animals  under  exper- 
iment," University  of  Penna.  Med.  Bull.,  1903,  No.  2,  p.  78. 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS.    239 

the  clamp  opened  the  anterior  and  posterior  wire  of  each 
pair  falls  away  from  the  median  line,  thereby  liberating 
the  animal.  To  secure  the  animal  it  is  placed  upon  its 
back,  the  head  laid  in  the  cradle  formed  by  the  bent 
wires,  the  latter  are  adjusted  to  the  proper  position,  and 
all  secured  by  the  turn  of  the  single  set-screw.  Of 
course,  the  extremities  of  the  animal  are  to  be  secured. 
This  is  done  by  means  of  cords  securely  held  by  a 
patent  fastener  made  by  the  Tie  Co.,  of  Unadilla,  N.  Y. 
These  fasteners  are  in  every  way  more  convenient  than 
the  elects  in  common  use.  An  idea  of  the  apparatus  is 
given  in  Fig.  43. 

A  very  simple  and  useful  holder  for  guinea-pigs  con- 
sists of  a  metal  cylinder  of  about  5  cm.  diameter  and 
about  13  cm.  long,  closed  at  one  end  by  a  perforated 
cap  of  either  tin  or  wire  netting.  Along  the  side  of 
this  box  is  a  longitudinal  slit  12  mm.  wide  that  runs  for 
9.5  cm.  from  within  0.5  cm.  of  the  open  extremity  of 
the  cylinder.  The  animal  is  placed  in  such  a  cylinder 
with  its  head  toward  the  perforated  bottom.  It  is  then 
easily  possible  to  make  subcutaneous  inoculation  by 
taking  up  a  bit  of  skin  through  the  slit  in  the  side  of 
the  box,  or  to  make  intraperitoneal  injection  by  drawing 
the  posterior  extremities  slightly  from  the  box  and 
holding  them  steady  between  the  index  and  second 
finger,  as  seen  in  Fig.  44.  It  is  also  very  convenient 
for  use  when  the  rectal  temperature  of  these  small  ani- 
mals is  to  be  taken.  The  manipulations  can  easily  be 
made  without  the  aid  of  an  assistant.  Its  construction 
is  seen  in  Fig.  44.1 

For  ordinary  subcutaneous   inoculations  at  the  root 

1  Centralblatt  fur  Bacteriologie  und  Parasitenkunde,  1895,  vol.  xviii. 
p.  530. 


240 


BACTERIOLOGY. 


of  the  tail  in  mice  a  simple  apparatus  consists  of  a 
piece  of  board  about  7  x  10  cm.  and  2  cm.  thick, 
upon  which  is  tacked  a  hollow  truncated  cone  of  wire 
gauze,  about  6  cm.  long  and  about  1.5  cm.  in  diameter 
at  one  end  and  2  cm.  at  its  other  end.  This  is  tacked 

FIG.  44. 


The  Voges  holder  for  guinea-pigs. 


upon  the  board  in  such  a  position  that  its  long  axis  is 
in  the  long  axis  of  the  board,  being  equidistant  from  its 
sides.  Its  small  end  is  placed  at  the  edge  of  the  board. 
The  mouse  is  taken  up  by  the  tail  by  means  of  a  pair 


INJECTION  INTO   THE  CIRCULATION.         241 

of  tongs  and  allowed  to  crawl  into  the  smaller  end  of 
the  wire  cone.  When  so  far  in  that  only  the  root  of 
the  tail  projects  the  animal  is  fixed  in  this  position  by 
a  clamp  and  thumb-screw,  with  which  the  apparatus 
(Fig.  45)  is  provided.  The  animal  usually  remains  per- 
fectly quiet  and  may  be  handled  without  difficulty. 

The  hair  over  the  root  of  the  tail  is  to  be  care- 
fully cut  away  with  scissors  and  a  pocket  cut  through 
the  skin  at  this  point.  The  inoculation  is  then  made 
into  the  loose  tissue  under  the  skin  over  this  part  of  the 
back  in  the  way  that  has  just  been  described.  It  is 
always  best  to  insert  the  needle  some  distance  along  the 

FIG.  45. 


Mouse-holder,  with  mouse  in  proper  position. 

spinal  column,  and  thus  deposit  the  material  as  far  from 
the  surface-wound  as  possible. 

As  the  subcutaneous  inoculation  is  very  simple  and 
takes  only  a  few  moments,  guinea-pigs,  rabbits,  and 
pigeons  may  be  held  by  an  assistant.  The  front  legs  in 
the  one  hand  and  the  hind  legs  in  the  other,  with  the 
animal  stretched  upon  its  back  on  a  table,  is  the  usual 
position  for  the  operation  when  practised  upon  guinea- 
pigs  and  rabbits.  The  point  at  which  the  inoculations 
are  commonly  made  is  in  the  abdominal  wall,  either  to 
the  right  or  left  of  the  median  line  and  about  3  cm. 

16 


242  BACTERIOLOGY. 

distant.  When  pigeons  are  used  they  are  held  with  the 
legs,  tail,  and  ends  of  the  wings  in  the  one  hand,  and 
the  head  and  anterior  portion  of  the  body  in  the  other, 
leaving  the  area  occupied  by  the  pectoral  muscles,  over 
which  the  inoculation  is  to  be  made,  free  for  manipu- 
lation. In  the  case  of  fur-bearing  animals  the  hair  over 
the  point  selected  for  the  inoculation  should  be  closely 
cut  with  scissors,  and  from  a  small  area  the  feathers 
should  be  plucked  in  the  case  of  birds. 

INJECTION  INTO  THE  CIRCULATION. — It  is  not  in- 
frequently desirable  to  inject  the  material  under  consid- 
eration directly  into  the  circulation  of  an  animal.  If 
a  rabbit  is  employed  for  the  purpose,  the  operation  is 
usually  done  upon  one  of  the  veins  in  the  ear.  To  those 
who  have  had  no  practice  with  this  procedure  it  offers  a 
great  many  difficulties ;  but  if  the  directions  which  will 
be  given  are  strictly  observed,  the  greatest  of  these 
obstacles  to  the  successful  performance  of  the  operation 
may  be  overcome. 

When  viewing  the  circulation  in  the  ear  of  the  rabbit 
by  transmitted  light  three  conspicuous  branches  of  the 
main  vessel  (vena  auricularis  posterior)  will  be  seen. 
One  runs  about  centrally  in  the  long  axis  of  the  ear, 
one  runs  along  its  anterior  margin,  and  one  along  its 
posterior  margin.  The  central  branch  (ra/mus  anterior 
of  the  vena  auricularis  posterior)  is  the  largest  and  most 
conspicuous  vessel  of  the  ear,  and  is,  therefore,  believed 
by  the  inexperienced  to  be  the  branch  into  which  it  would 
appear  easiest  to  insert  a  hypodermic  needle.  This, 
however,  is  fallacious.  This  vessel  lies  very  loosely 
imbedded  in  connective  tissue,  and,  in  eiforts  to  intro- 
duce a  needle  into  it,  rolls  about  to  such  an  extent  that 
only  after  a  great  deal  of  difficulty  does  the  experiment 
succeed.  On  the  other  hand,  the  posterior  branch  (ramus 


INJECTION  INTO  THE  CIRCULATION.          243 

later  alls  posterior  of  the  vena  auricularis  posterior)  is  a 
very  fine,  delicate  vessel  which  runs  along  the  posterior 
margin  of  the  ear,  and  is  so  firmly  fixed  in  the  dense 
tissues  which  surround  it  that  it  is  prevented  from 
rolling  about  under  the  point  of  the  needle.  The 
further  away  from  the  mouth  of  the  vessel — that  is,  the 
nearer  we  approach  its  capillary  extremity — the  more 
favorable  become  the  conditions  for  the  success  of  the 
operation. 

After  shaving  the  ear  and  carefully  washing  it  with 
clean  water  select  the  very  delicate  vessel  lying  quite 
close  to  the  posterior  margin  of  the  ear,  and  make  the 
injection  as  near  to  the  apex  of  the  ear  as  possible.  At 
times  the  vessels  of  the  ear  will  be  found  to  contain 
so  little  blood  that  they  are  hardly  distinguishable, 
making  it  very  difficult  to  introduce  the  needle  into 
them.  This  is  sometimes  overcome  by  pressure  at  the 
root  of  the  ear,  causing  stasis  of  the  blood  and  distention 
of  the  vessels.  A  very  satisfactory  method  of  causing 
the  veins  to  become  prominent  is  to  press  lightly  or 
prick  gently  with  the  point  of  a  needle  the  skin  over 
the  vessel  to  be  used.  In  a  few  seconds,  as  a  result  of 
this  irritation,  the  vessel  will  have  become  distended 
with  blood,  and  readily  distinguishable  from  the  sur- 
rounding tissue;  it  may  then  be  easily  punctured  by 
the  needle  of  the  syringe.  A  sharp  flick  with  the 
finger  will  often  produce  the  same  result.  The  injection 
is  always  to  be  made  from  the  dorsal  surface  of  the  ear. 

Of  no  less  importance  than  the  selection  of  the  proper 
vessel  is  the  shape  of  the  point  of  the  needle  employed. 
The  hypodermic  needles  as  they  come  from  the 
makers  are  not  suited  at  all  for  this  operation,  because 
of  the  manner  in  which  their  points  are  ground.  If 
one  examine  carefully  the  point  of  a  new  hypodermic 


244  BACTERIOLOGY. 

needle,  it  will  be  seen  that  the  long  point,  instead  of 
presenting  a  flat,  slanting  surface  when  viewed  from  the 
side,  has  a  more  or  less  curved  surface.  Now,  in  efforts 
to  introduce  such  a  needle  into  a  vessel  of  very  small 
calibre  it  is  usually  seen  that  the  point  of  the  needle, 
instead  of  remaining  in  the  vessel,  as  it  would  do  were 
it  straight  (or  "  chisel  pointed "),  very  commonly  pro- 
jects into  the  opposite  wall ;  and  as  the  needle  is  inserted 
further  and  further  it  is  usually  pushed  through  the 
vessel-walls  into  the  loose  tissues  beyond,  and  the 
material  to  be  injected  is  deposited  in  these  tissues, 
instead  of  into  the  circulation.  If,  on  the  contrary, 
the  slanting  point  of  the  needle  be  ground  until  its  sur- 
face is  perfectly  flat  when  viewed  from  the  side,  and  no 
curvature  exists,  then  when  once  inserted  it  usually 
remains  within  the  vessel,  and  there  is  no  tendency  to 
penetrate  the  opposite  wall.  AVe  never  use  a  new  hypo- 
dermic needle  until  its  point  is  carefully  ground  to  a  per- 
fectly flat,  slanting  surface  with  no  curvature  whatever. 
These  differences  may  perhaps  be  more  easily  under- 
stood if  represented  diagrammatically.  In  Fig.  46,  a, 

FIG.  46. 
a 


Hypodermic  needles,  magnified,  a.  Improper  point.  6.  Proper  shape  of  point. 

the  needle  has  the  point  usually  seen  when  new.  In  Fig. 
47,  6,  the  point  has  been  ground  to  the  shape  best  suited 
for  this  operation.  The  needles  need  not  be  returned 
to  the  maker.  One  can  grind  them  to  the  shape  desired 
in  a  few  minutes  upon  an  oilstone.  The  size  of  the 


INJECTION  INTO   THE  CIRCULATION.          245 

needle   is  that  commonly  employed   by  physicians  for 
subcutaneous  injections  in  human  beings. 

When  the  operation  is  to  be  performed  an  assistant 
holds  the  animal  gently  but  firmly  in  the  crouching 
position  upon  a  table.  If  the  animal  does  not  remain 
quiet,  it  is  best  to  wrap  it  in  a  towel,  so  that  only  its 
head  protrudes;  though  in  most  cases  we  have  not 
found  this  necessary,  particularly  if  the  animal  has  not 
been  excited  prior  to  beginning  the  operation. 

The  ear  in  which  the  injection  is  to  be  made  should  be 
shaved  clean  of  hair  by  means  of  a  razor  and  soap  and 
then  washed  with  water.  It  is  unnecessary  to  attempt 
disinfection  of  the  skin. 

The  animal  should  be  placed  so  that  the  prepared 
ear  comes  between  the  operator  and  the  source  of  light. 
This  renders  visible  by  transmitted  light  not  only  the 
coarser  vessels  of  the  ear,  but  also  their  finer  branches. 

The  filled  hypodermic  syringe  is  taken  in  one  hand 
and  with  the  other  hand  the  ear  is  held  firmly.  The 
point  of  the  needle  is  then  inserted  through  the  skin 
and  into  the  finest  part  of  the  ramus  posteriory  the  part 
nearest  the  apex  of  the  ear,  where  the  course  of  the 
vessel  is  nearly  straight.  When  the  point  of  the  needle 
is  in  this  vessel  it  gives  to  the  hand  a  sensation  quite 
different  from  that  felt  when  it  is  in  the  midst  of  con- 
nective tissue.  As  soon  as  one  supposes  the  point  of 
the  needle  is  in  the  vessel  a  drop  or  two  of  the  fluid  may 
be  injected  from  the  syringe,  and,  if  his  suspicions  are 
correct,  the  circulation  in  the  small  ramifications  and 
their  anastomoses  will  rapidly  alter  in  appearance — 
i.  e.,  the  circulating  blood  will  be  displaced  very  quickly 
by  the  clear,  transparent  fluid  that  is  being  injected.  At 
this  stage  one  must  proceed  very  carefully,  for  some- 
times when  the  needle-point  is  not  actually  in  the  ves- 


246  BA  CTERTQLOG  Y. 

sel,  but  is  in  the  lymph-spaces  surrounding  it,  an  ap- 
pearance somewhat  similar  is  seen.  This  may  always 
be  differentiated,  however,  by  continuing  the  injection, 
when  the  flow  of  clear  fluid  through  the  vessels  will  not 
only  fail  to  take  the  place  of  the  circulating  blood,  but 
at  the  same  time  a  localized  swelling,  due  to  an  accu- 
mulation of  the  fluid  injected,  will  appear  under  the 
skin  about  the  point  of  the  needle.  The  needle  must 
then  be  withdrawn  and  inserted  into  the  vessel  at  a 
point  a  little  nearer  its  proximal  end. 

Care  must  be  taken  that  no  air  is  injected. 

The  hypodermic  syringe  and  needle  must,  previous 
to  operation,  have  been  carefully  sterilized  in  the  steam 
sterilizer  or  in  boiling  water.  The  animal  must  be  kept 
under  close  observation  for  about  an  hour  after  injection. 

The  operation  is  one  that  cannot  be  learned  from 
verbal  description.  It  can  only  be  successfully  per- 
formed after  actual  practice.  If  the  precautions  which 
have  been  mentioned  are  observed,  but  little  difficulty 
in  performing  the  operation  will  be  experienced. 

Its  greater  convenience  and  simplicity,  as  compared 
with  other  methods  for  the  introduction  of  substances 
into  the  circulation,  commend  it  as  a  technical  procedure 
with  which  to  make  one's  self  familiar.  The  animals 
sustain  practically  no  wound,  they  experience  no  suffer- 
ing— at  least  they  give  no  evidence  of  pain — and  no 
anaesthetic  is  required. 

The  form  of  syringe  best  suited  for  this  operation  is 
of  the  ordinary  design,  but  one  that  permits  of  thorough 
sterilization  by  steam.  It  should  be  made  of  glass  and 
metal,  with  packings  that  may  be  sterilized  by  steam 
without  injury.  The  syringes  commonly  employed  are 
those  shown  in  Fig.  47. 

For   operations   requiring   exact   dosage    experience 


INOCULATION  INTO  LYMPHATIC  CIRCULATION.  247 

has  led  me  to  prefer  a  syringe  after  the  pattern  of  C, 
in  Fig.  47 — i.  e.j  the  form  commonly  used  by  physi- 
cians. The  reason  for  this  is  as  follows :  in  making 
injections,  either  into  the  circulation  or  under  the  skin, 
there  is  a  certain  amount  of  resistance  to  the  passage  of 
fluid  from  the  needle.  If  one  overcomes  this  resistance 
by  means  of  a  cushion  of  compressed  air,  as  is  the  case 
in  syringes  A  and  j3,  Fig.  47,  the  sudden  expansion 
of  the  air  in  the  body  of  the  syringe  when  resistance  is 
overcome  frequently  causes  a  larger  amount  of  fluid  to 
be  injected  than  is  desired.  No  such  accident  is  likely 

FIG.  47. 


Forms  of  hypodermic  syringe. 
A.  Koch's  syringe.    B.  Syringe  of  Strohschein.    C.  Overlack's  form. 

to  occur  when  the  fluid  is  forced  from  the  barrel  of  the 
syringe  by  the  head  of  a  close-fitting  piston,  with  no  air 
intervening  between  the  fluid  and  the  head  of  the  piston. 
With  such  an  instrument,  properly  manipulated,  the 
dose  can  always  be  controlled  with  accuracy. 

INOCULATION  INTO  THE  LYMPHATIC  CIRCULATION. 
— Fluid  cultures  or  suspensions  of  bacteria  may  be  in- 
jected into  the  lymphatics  by  way  of  the  testicles.  The 
operation  is  in  no  wise  complicated.  One  simply  plunges 
the  point  of  the  hypodermic  needle  directly  into  the  sub- 


248  BACTERIOLOGY. 

stance  of  the  testicle  and  then  injects  the  amount  desired. 
Injections  made  in  this  manner  are  sometimes  followed 
by  interesting  pathological  lesions  of  the  lymphatic 
apparatus  of  the  abdomen. 

INOCULATION  INTO  THE  GREAT  SEROUS  CAVITIES. 
— Inoculation  into  the  peritoneum  presents  no  difficulties 
if  fluids  are  to  be  introduced.  In  this  case  one  makes, 
with  a  pair  of  sterilized  scissors,  a  small  nick  through 
the  skin  down  to  the  underlying  fasciae,  and,  taking  a 
fold  of  the  abdominal  wall  between  the  fingers,  plunges 
the  hypodermic  needle  through  the  opening  just  made 
directly  into  the  peritoneal  cavity.  There  is  little  or  no 
danger  of  penetrating  the  intestines  or  other  internal 
viscera  if  the  puncture  be  made  along  the  median  line 
at  about  midway  between  the  end  of  the  sternum  and 
the  symphysis  pubis.  Though  this  may  seem  a  rude 
method,  it  is  rare  that  the  intestines  are  penetrated  or 
otherwise  injured.  The  object  of  the  primary  incision 
is  to  lessen  the  chances  of  contamination  by  bacteria 
located  in  the  skin,  some  of  which  might  adhere  to  the 
needle  if  it  were  plunged  directly  through  the  skin,  and 
thus  complicate  the  results. 

If  solid  substances,  bits  of  tissue,  etc.,  are  to  be  intro- 
duced into  the  peritoneum,  it  becomes  necessary  to  con- 
duct the  operation  upon  the  lines  of  a  laparotomy. 
The  hair  should  be  shaved  from  a  small  area  over  the 
median  line,  after  which  the  skin  is  to  be  thoroughly 
washed.  A  short  longitudinal  incision  (about  2  cm. 
long)  is  then  to  be  made  in  the  median  line  through  the 
skin  and  down  to  the  fasciae.  Two  subcutaneous 
sutures,  as  employed  by  Halsted,  are  then  to  be  intro- 
duced transversely  to  the  line  of  incision  about  1  cm. 
apart,  and  their  ends  left  loose.  This  particular  sort  of 
suture  does  not  pass  through  the  skin,  but,  instead,  the 


INOCULATION  INTO   GREAT  SEROUS  CAVITIES.  249 

needle  is  introduced  into  the  subcutaneous  tissues  along 
the  edge  of  the  incision.  In  this  case  they  are  to  pass 
into  the  abdominal  cavity  and  out  again,  entering  at  one 
side  of  the  line  of  incision  and  leaving  at  the  other, 
as  indicated  by  the  solid  and  dotted  lines  in  Fig.  48. 
(The  figure  indicates  the  primary  opening  through  the 
skin.  The  longitudinal  dotted  line  shows  the  opening  to 

FIG.  48. 


Diagram  of  skin  incision  and  sutures  in  laparotomy  on  animals. 

be  made  into  the  abdomen ;  the  transverse  dotted  lines, 
with  their  loose  ends,  represent  the  sutures  as  placed 
in  position  before  the  abdomen  is  opened  ;  it  will  be  seen 
that  these  sutures  in  all  cases  pass  through  the  subcuta- 
neous tissues  only  and  do  not  penetrate  the  skin  proper.) 
The  opening  through  the  remaining  layers  may  now 
be  completed ;  the  bit  of  tissue  is  deposited  in  the  peri- 
toneal cavity,  under  precautions  that  will  exclude  all 
else,  the  edges  of  the  wound  drawn  evenly  and  gently 
together  by  tying  the  sutures,  and  the  lines  of  incision 
dressed  with  collodion.  It  should  be  needless  to  say 
that  this  operation  must  be  conducted  under  the  strictest 


250  BACTERIOLOGY. 

precautions,  to  avoid  complications.  All  instruments, 
sutures,  ligatures,  etc.,  must  be  carefully  sterilized  either 
in  the  steam  sterilizer  for  twenty  minutes,  or  by  boiling 
in  2  per  cent,  sodium  carbonate  solution  for  ten  min- 
utes ;  the  hands  of  the  operator,  though  they  should  not 
touch  the  wound,  must  be  carefully  cleansed,  and  the 
material  to  be  introduced  into  the  abdomen  should  be 
handled  with  only  sterilized  instruments. 

Inoculation  into  the  pleural  cavity  is  much  less  fre- 
quently required — in  fact,  it  is  not  a  routine  method. 
It  is  not  easy  to  enter  the  pleural  cavity  with  a  hypo- 
dermic needle  without  injuring  the  lung,  and  it  is  rare 
that  conditions  call  for  the  introduction  of  solid  parti- 
cles into  this  locality. 

Inoculation  into  the  anterior  chamber  of  the  eye  is  per- 
formed by  making  a  puncture  through  the  cornea  just 
in  front  of  its  junction  with  the  sclerotic,  the  knife  being 
passed  into  the  anterior  chamber  in  a  plane  parallel  to 
the  plane  of  the  iris.  By  the  aid  of  a  fine  pair  of  for- 
ceps the  bit  of  tissue  is  passed  through  the  opening  thus 
made  and  is  deposited  upon  the  iris,  where  it  is  allowed 
to  remain,  and  where  its  pathogenic  activities  upon  the 
iris  can  be  conveniently  studied.  It  is  a  mode  of  inoc- 
ulation of  very  limited  application,  and  is  therefore  but 
rarely  practised.  It  was  employed  in  the  classical 
experiments  of  Cohnheim  in  demonstrating  the  infec- 
tious nature  of  tuberculous  tissues,  tuberculosis  of  the 
iris  being  the  constant  result  of  the  introduction  of  tuber- 
culous tissue  into  the  anterior  chamber  of  the  eye  of  rabbits. 

OBSERVATION  OF  ANIMALS  AFTER  INOCULATION. 
— After  either  of  these  methods  of  inoculation,  particu- 
larly when  unknown  species  of  bacteria  are  being  tested, 
the  animal  is  to  be  kept  under  constant  observation  and 
all  deviations  from  the  normal  are  to  be  carefully  noted 


AMMALS  Al'TKll   INOCULATION. 


251 


— as,    for   instance,  elevation   of   temperature  ;    loss  of 
weight  ;   peculiar  position  in  the  cage  ;   loss  of  appetite  ; 


•Qt 

s 

H 

s 

M 

. 

H 

u 

k 

01 

7 

e 

^ 

8 

i 

L 

VN 

i 

0 

s 

0 

^ 

K 

V 

V, 

i 

1 

> 

w 

'•~ 

2 

--.  ^ 

J 

-. 

t 

X 

IE 

S 

oe 

j 

H 

> 

f 

M 

,^v 

1 

/z 

•'" 

S 

92 

. 

& 

52 

^> 

| 

« 

X 

n 

1 

zz 

j 

IZ 

^ 

1 

oz 

^ 

j 

8t 

s 

& 

21 

f 

S 

LI 

,  J 

91 

\_ 

91 

J 

1 

M 

^ 

1 

', 

n 

• 

^ 

i 

4 

'^ 

Zl 

< 

I 

7, 

11. 

r> 

v 

;!»M 

I 

:-; 

1 

2 

• 
•» 

«-„. 

j-1-.j     < 

Si 

- 

I 


1-1 


a  c- 


roughening  of  the  hair;  exeessive  seeretions,  from  eitli«;r 
the  air-pa--;iL!'  -. '•'•njiirx-tiva,  or  kidneys;  IOO-<-IH-—  of  or 
hemorrhage  from  the  bowels;  tumefaction  or  reaction  at 


252 


BACTERIOLOGY. 


site  of  inoculation,  etc.     If  death  ensue  in  from  two  to 
four  days,  it  may  reasonably  be  expected  that  at  autopsy 


SI 

f 

*l 

i 

et 

El 

11 

i 

01 

*"<* 

6 

^ 

> 

8 

f 

L 

s 

9 

' 

•3 

s 

^ 

^ 

5 

t 

"7 

g 

April  ,1394. 

e 
z 

•E 

;==  • 

:  =  =  = 

-  -  = 
=  -- 

—  —  • 

=  =; 

-  « 

-* 

-- 

-  «. 

'  Not  less  th 

18 

oe 

\ 

**  " 

6Z 

s 

1 

83 

V 

1 

/z 

.~> 

I 

92 

j 

.-' 

6. 

23 
fro 

1 

•  - 

—  •—  • 

=  — 

e  = 

-     J 

EC 

s 

23 

' 

y  x 

1 

13 

~e 

^> 

1 

03 

•- 

C 

a 

61 

" 

"5 

| 

81 

i 

§ 

a 

'„ 

x  * 

9V 

' 

J 

^^ 

SI 

[ 

^, 

V 

3 

H 

'- 

-  - 

—  ^* 

•^ 

i 

i 

£1 

^ 

7< 

—• 

i 

1 

3-1 

N 

> 

& 

1 

U 

V 

»M 

»!»M 

1 

f 

! 

S 

i 

! 

g 

SJIHH 

"^'X 

5 

s 

S 

• 

evidence  of  either  acute  septic  or  toxic  processes  will  be 
found.  It  sometimes  occurs,  however,  that  inoculation 
results  in  the  production  of  chronic  conditions,  and  the 


ANIMALS  AFTER  INOCULATION. 


253 


animal  must  be  kept  under  observation  often  for  weeks. 
In  these  cases  it  is  important  to  note  the  progress  of 


•Ql 

I  s 

U                 n 



i 

El 

5 

31 

'c/. 

u          ; 

'> 

01               e 

- 

6 

—  • 

8                     r 

^ 

g 

Z                 r' 

o 

0                 J 

» 

S                1 

f  x              1 

'E. 

•fr                \ 

5 

t 

3 

e           c 

.  _  /  _  ? 

c 

2 

z        <;' 

tr. 

< 

i          * 

^                                  H 

I 

te 

g 

0&                o 

* 

63 

:;:  t 

^ 

83 

'ti. 

18 

__ 

* 

93 

i 

'C 

S3 

/     § 

5 

•*2 

c 

£3 

| 

33             ' 

)     t 

8, 

13 

.  _  .    •§ 

OS          <;' 

61 

^     i 

a: 

81 

§ 

§ 

n.         ' 

^ 

M 

91 

1 

Ql 

i 

£ 

H                I 

s 

00 

I 

El 

i 

i 

s 

31 

^>       .& 

3 

1 

U                                         * 

§ 

or. 

• 

3!"AV.                                           ? 

g     § 

.«,. 

"*—i    SSSSS 

the  disease  by  its  effect  upon  the  physical  condition  of 
the  animal,  viz.,  upon  the  nutritive  processes,  as  evi- 


254 


BACTERIOLOGY. 


denced  by  fluctuation  in  weight,  and  upon  the  body- 
temperature.     For  this    purpose    the    animal    is  to  be 


weighed  daily,  always   at  about  the   same  hour  and 
always  about  midway  between  the  hours  of  feeding  \ 


ANIMALS  AFTER  INOCULATION.  255 

at  the  same  time  its  temperature,  as  indicated  by  a 
thermometer  placed  in  the  rectum,  is  to  be  recorded.1 
By  comparison  of  these  daily  observations  the  ob- 
server is  aided  in  determining  the  course  the  infection 
is  taking. 

Too  much  stress  must  not,  however,  be  laid  upon 
moderate  and  sudden  daily  fluctuations  in  either  tem- 
perature or  weight,  as  it  is  a  common  observation  that 
presumably  normal  animals  when  confined  in  cages  and 
fed  regularly  often  present  very  striking  temporary 
gains  and  losses  in  weight,  often  amounting  to  50  or 
100  grammes  in  twenty-four  hours,  even  in  animals 
whose  total  weight  may  not  exceed  500  or  600  gammes ; 
similarly  unexplai liable  rises  and  falls  of  temperature, 
often  as  much  as  a  degree  from  one  day  to  another,  are 
seen.  Such  fluctuations  have  apparently  no  bearing 
upon  the  general  condition  of  the  animal,  but  are  prob- 
ably clue  to  transient  causes,  such  as  overfeeding  or 
scarcity  of  food,  improper  feeding,  lack  of  exercise, 
excitement,  fright,  etc. 

The  accompanying  charts  (Figs.  49,  50,  51,  52)  will 
serve  to  illustrate  some  of  these  points.  The  animals, 
two  rabbits  and  two  guinea-pigs,  were  taken  at  random 
from  among  stock  animals  and  placed  each  in  a  clean 
cage,  the  kind  used  for  animals  under  experiment,  and 
kept  under  as  good  general  conditions  as  possible.  For 
the  first  week  the  rabbits  received  each  100  grammes 
of  green  food  (cabbage  and  turnips)  daily,  and  the 
guinea-pigs  30  grammes  each  of  the  same  food.  During 


1  The  thermometer  must  be  inserted  into  the  rectum  beyond  the 
grasp  of  the  sphincter,  otherwise  pressure  upon  its  bulb  by  contraction 
of  this  muscle  may  force  up  the  mercurial  column  to  a  point  higher 
than  that  resulting  from  the  actual  body-temperature. 


2  56  BA  CTERIOL  OGY. 

the  second  week  this  daily  amount  of  food  was  doubled  ; 
during  the  third  week  it  was  quadrupled ;  and  for  the 
fourth  and  fifth  weeks  they  each  received  an  excess  of 
food  daily,  consisting  of  green  vegetables  and  grains 
(oats  and  corn).  By  reference  to  the  charts  sudden 
diurnal  fluctuations  in  weight  will  be  observed  that 
do  not  correspond  in  all  instances  with  scarcity  or  suf- 
ficiency of  food.  With  the  rabbits  there  is  a  gradual 
loss  of  weight  with  the  smaller  amounts  of  food,  which 
losses  are  not  totally  recovered  as  the  food  is  increased. 
With  the  guinea-pigs  there  is  likewise  at  first  a  loss ; 
but  after  a  short  time  the  weight  remains  tolerably  con- 
stant, and  is  not  so  conspicuously  affected  by  the  increase 
in  food  as  one  might  expect.  From  the  recorded  tem- 
peratures one  sees  the  peculiar  fluctuations  mentioned. 
To  just  what  they  are  due  it  is  impossible  to  say.  It 
is  manifest  that  the  normal  temperature  of  these  animals, 
if  we  can  speak  of  a  normal  temperature  for  animals 
presenting  such  fluctuations,  is  about  a  degree  or  more, 
Centigrade,  higher  than  that  of  human  beings.  The 
animals  from  which  these  charts  were  made  were  not 
inoculated,  nor  were  they  subjected  to  any  operative 
procedures  whatever,  the  only  deviations  from  normal 
conditions  being  the  variations  in  the  daily  amount  of 
food  given. 

In  certain  instances,  however,  there  will  be  noticed 
a  constant  tendency  to  diminution  in  weight,  notwith- 
standing the  daily  fluctuations,  and  after  a  time  a  con- 
dition of  extreme  emaciation  may  be  reached,  the 
animal  often  being  reduced  to  from  50  to  60  per  cent, 
of  its  original  weight.  In  other  cases,  after  inoculations 
to  which  the  animal  is  not  susceptible,  rabbits  in  par- 
ticular, if  properly  fed,  will  frequently  gain  steadily  in 


ANIMALS  AFTER   INOCULATION.  257 

weight.  The  condition  of  progressive  emaciation  just 
mentioned  is  conspicuously  seen  after  intravenous  inoc- 
ulation of  rabbits  with  cultures  of  bacillus  typhosus  and 
of  bacillus  coli,  referred  to  in  the  chapter  on  the  latter 
organism,  and  if  looked  for  will  doubtless  be  seen  to  fol- 
low inoculation  with  other  organisms  capable  of  producing 
chronic  forms  of  infection,  but  which  are  frequently  con- 
sidered non-pathogenic  because  of  their  inability  to  induce 
acute  conditions.  Not  infrequently  in  chronic  infections 
there  may  be  hardly  any  marked  and  constant  temperature- 
variations  until  just  before  death,  when  sometimes  there 
will  be  a  rise  and  at  other  times  a  fall  of  temperature. 
In  the  majority  of  cases,  however,  one  must  be  very 
cautious  as  to  the  amount  of  stress  laid  upon  changes 
in  weight  and  temperature,  for  unless  they  are  progres- 
sive or  continuous  in  one  or  another  direction  they  may 
have  little  or  no  significance  as  indicating  the  existence 
or  absence  of  disease. 

17 


CHAPTER    XIII. 

Post-mortem  examination  of  animals — Bacteriological  examination  of 
the  tissues — Disposal  of  tissues  and  disinfection  of  instruments 
after  the  examination — Study  of  tissues  and  exudates  during  life. 

DURING  bacteriological  examination  of  the  tissues 
of  dead  animals  certain  precautions  must  be  rigidly 
observed  in  order  to  arrive  at  correct  conclusions. 

The  autopsy  should  be  made  as  soon  as  possible 
after  death.  If  delay  cannot  be  avoided,  the  animal 
should  be  kept  on  ice  until  the  examination  can  be 
made,  otherwise  decomposition  sets  in,  and  the  sapro- 
phytic  bacteria  now  present  may  interfere  with  the 
accuracy  of  results.  When  the  autopsy  is  to  be  made 
the  animal  is  first  inspected  externally,  and  all  visible 
lesions  noted.  It  is  then  to  be  fixed  upon  its  back 
upon  a  board  with  nails  or  tacks.  The  four  legs  and 
the  end  of  the  nose,  through  which  the  tacks  are  driven, 
are  to  be  moderately  extended.  Plates  are  now  to  be 
made  from  the  site  of  inoculation,  if  this  is  subcuta- 
neous. The  surfaces  of  the  thorax  and  abdomen  are 
then  to  be  moistened  to  prevent  the  fine  hairs,  dust, 
etc.,  from  floating  about  in  the  air  and  interfering  with 
the  work.  An  incision  is  then  made  through  the  skin 
from  the  chin  to  the  symphysis  pubis.  This  is  only 
a  skin  incision,  and  does  not  reach  deeper  than  the 
muscles.  It  is  best  done  by  first  making  with  a  scalpel 
an  incision  just  large  enough  to  permit  of  the  intro- 
duction of  one  blade  of  a  blunt-pointed  scissors.  It  is 

258 


POST-MORTEM  EXAMINATION  OF  ANIMALS.   259 

then  completed  with  the  scissors.  The  whole  of  the 
skin  is  now  to  be  carefully  dissected  away,  not  only 
from  the  abdomen  and  thorax,  but  from  the  axillary, 
inguinal,  and  cervical  regions,  and  the  fore  and  hind 
legs  as  well.  It  is  then  pinned  flat  upon  the  board 
so  as  to  keep  it  as  far  from  the  abdomen  and  thorax  as 
possible,  for  it  is  from  the  skin  that  the  chances  of 
contamination  are  greatest. 

It  now  becomes  necessary  to  proceed  very  carefully. 
All  incisions  from  this  time  on  are  to  be  made  only 
through  surfaces  that  have  been  sterilized.  The  sterili- 
zation is  best  accomplished  by  the  use  of  a  broad-bladed 
table-knife  that  has  been  heated  in  a  gas-flame.  The 
blade,  made  quite  hot,  is  to  be  held  upon  the  region  of 
the  linea  alba  until  the  tissues  of  that  region  begin  to 
burn  ;  it  is  then  held  transversely  to  this  line  over  about 
the  centre  of  the  abdomen,  thus  making  two  sterilized 
tracks,  through  which  the  abdomen  may  be  opened  by 
a  crucial  incision.  The  sterilization  thus  accomplished 
is,  of  course,  directed  only  against  organisms  that  may 
have  fallen  upon  the  surface  from  without,  and  there- 
fore it  need  not  extend  deep  down  through  the  tissues. 
In  the  same  way  two  burned  lines  may  be  made  from 
either  extremity  of  the  transverse  line  up  to  the  top  of 
the  thorax. 

With  hot  scissors  the  central  longitudinal  incision 
extending  from  the  point  of  the  sternum  to  the  geni- 
talia  is  to  be  made  without  touching  the  internal  vis- 
cera. The  abdominal  wall  must  therefore  be  held  up 
during  the  operation  with  sterilized  forceps  or  hooks. 
The  cross-incision  is  made  in  the  same  way.  When 
this  is  completed  an  incision  through  the  ribs  with  a 
pair  of  heavy,  sterilized  scissors  is  made  along  the 


260  BA  (JTERIOLOG  Y. 

scorched  tracks  on  either  side  of  the  thorax.  After  this 
the  whole  anterior  wall  of  the  thorax  may  easily  be 
lifted  up,  and  by  severing  the  connections  with  the 
diaphragm  it  may  be  completely  removed.  When  this 
is  done  and  the  abdominal  flaps  laid  back,  the  contents 
of  both  cavities  are  to  be  inspected  and  their  condition 
noted  without  disturbing  them. 

After  this  the  first  steps  to  be  taken  are  to  prepare 
plates  or  Esmarch  tubes  from  the  blood,  liver,  spleen, 
kidneys,  and  any  exudates  that  may  exist.  This  is  best 
done  as  follows :  Heat  a  scalpel  quite  hot  and  apply  it 
to  a  small  surface  of  the  organ  from  which  cultures  are 
to  be  made.  Hold  it  upon  the  organ  until  the  surface 
directly  beneath  is  visibly  scorched.  Then  remove  it, 
heat  it  again,  and  while  quite  hot  insert  its  point  through 
the  capsule  of  the  organ.  Into  the  opening  thus  made 
insert  a  sterilized  platinum  loop,  made  of  wire  a  little 
heavier  than  that  commonly  employed.  Project  this 
deeply  into  the  tissues  of  the  organ  ;  by  twisting  it 
about  enough  material  from  the  centre  of  the  organ 
can  be  obtained  for  making  the  cultures. 

As  the  resistance  offered  by  the  tissue  is  sometimes 
too  great  to  permit  of  puncture  with  the  ordinary  wire 
loop,  Nuttall l  has  devised  for  the  purpose  a  platinum- 
wire  spear  which  possesses  great  advantage  over  the 
loop.  It  has  the  form  seen  in  Fig.  53.  It  is  easily 
made  by  beating  a  piece  of  heavy  platinum  wire  into  a 
spear-head  at  one  end,  and  perforating  this  with  a  small 
drill,  as  seen  in  the  cut.  It  is  attached  by  the  other  end 
to  either  a  metal  or  glass  handle,  preferably  the  former. 
It  can  readily  be  thrust  into  the  densest  of  the  soft  tissues, 

^entralbatt  fur  Bakteriologie  und  Parasjtenkunde,  1892,  Bd.  xi, 
p.  538, 


POST-MORTEM, EXAMINATION  OF  ANIMALS.    261 

and  by  twisting  it  about  after  its  introduction  particles 
of  the  tissue  sufficient  for  examination  are  withdrawn 
in  the  eye  of  the  spear-head. 


Nuttall's  platinum  spear  for  use  at  autopsies. 

Cultures  from  the  blood  are  usually  made  from 
one  of  the  cavities  of  the  heart,  which  is  always  punct- 
ured at  a  point  which  has  been  burned  in  the  way 
given. 

In  addition  to  cultures,  cover-slips  from  the  site  of 
inoculation,  from  each  organ,  and  from  any  exudates  that 
may  be  present  must  be  made.  These,  however,  are 
prepared  after  the  materials  for  the  cultures  have  been 
obtained.  They  need  not  be  examined  immediately, 
but  may  be  placed  aside,  under  cover,  on  bits  of  paper 
upon  which  the  name  of  the  organ  from  which  they  were 
prepared  is  written. 

When  the  autopsy  is  complete  and  the  gross  appear- 
ances have  been  carefully  noted,  small  portions  of  each 
organ  are  to  be  preserved  in  95  per  cent,  alcohol  for 
subsequent  examination.  Throughout  the  entire  au- 
topsy it  must  be  borne  in  mind  that  all  cultures, 
cover-slips,  and  tissues  must  be  carefully  labelled, 
not  only  with  the  name  of  the  organ  from  which 
they  originate,  but  with  the  date,  designation  of  the 
animal,  etc.,  so  that  an  account  of  their  condition 
after  closer  study  may  be  subsequently  inserted  in  the 
protocol. 

The  cover-slips  are  now  to  be  stained,  mounted,  and 


26!2  BA  CTERIOLOG  Y. 

examined  microscopically,  and  the  results  carefully 
noted. 

The  same  care  with  regard  to  noting,  labelling, 
etc.,  should  be  exercised  in  the  subsequent  study  of 
the  cultures  and  the  hardened  tissues,  which  are  to  be 
stained  and  subjected  to  microscopic  examination.  The 
results  of  microscopic  study  of  the  cover-slip  prepara- 
tions and  of  those  obtained  by  cultures  should  in  most 
cases  correspond,  though  it  not  rarely  occurs  that  bac- 
teria are  present  in  such  small  numbers  in  the  tissues 
that  their  presence  may  be  overlooked  microscopically, 
and  still  they  may  appear  in  the  cultures. 

If  the  autopsy  has  been  performed  in  the  proper 
way,  with  the  precautions  given,  and  sufficiently  soon 
after  death,  the  results  of  the  bacteriological  exami- 
nation should  be  either  negative  or  the  organisms 
which  are  isolated  should  be  in  pure  cultures.  This  is 
particularly  the  case  with  cultures  made  from  the  inter- 
nal viscera. 

Both  the  cover-slips  and  cultures  made  from  the 
point  of  inoculation  are  apt  to  contain  a  variety  of 
organisms. 

If  the  organism  obtained  in  pure  culture  from  the 
internal  viscera,  or  those  predominating  at  the  point  of 
inoculation  of  the  animal,  have  caused  its  death,  then 
subsequent  inoculation  of  pure  cultures  of  this  organism 
into  the  tissues  of  a  second  animal  should  produce  sim- 
ilar results. 

When  the  autopsy  is  quite  finished  the  remains  of 
the  animal  should  be  burned  ;  all  instruments  subjected 
to  either  sterilization  by  steam  or  boiling  for  fifteen 
minutes  in  a  1  to  2  per  cent,  soda  solution ;  and  the 
board  upon  which  the  animal  was  tacked,  as  well  as  the 


STUDIES   OF  TISSUES  DURING  LIFE.         263 

tacks,  towels,  dishes,  and  all  other  implements  used  at 
the  autopsy,  be  sterilized  by  steam.  All  cultures,  cover- 
slips,  and,  indeed,  all  articles  likely  to  have  infectious 
material  upon  them,  must  be  sterilized  as  soon  as  they 
are  of  no  further  service. 

What  has  been  said  with  regard  to  the  study  of  dead 
tissues  obtained  at  autopsy  applies  equally  well  to  the 
bacteriological  study  of  tissues  and  exudates  obtained 
during  life.  In  the  latter  case,  however,  certain  pre- 
cautions are  always  to  be  observed.  In  the  first  place, 
it  is  desirable  to  obtain  the  materials  under  aseptic  pre- 
cautions, care  being  taken  that  no  disinfectant  fluids 
are  mixed  with  them.  They  should  be  subjected  to 
study  as  soon  as  possible  after  removal  from  the  body. 
In  the  case  of  tissues  that  cannot  be  examined  on  the 
spot,  they  should  be  placed  in  a  sterile  Petri  dish  or 
in  a  stoppered,  sterile,  wide-mouthed  bottle  and  taken 
at  once  to  the  laboratory.  The  surface  should  then  be 
seared  with  a  hot  knife  and  an  incision  through  the 
seared  area  into  the  centre  made  with  a  knife  that  has 
been  sterilized  and  allowed  to  cool.  From  the  depths 
of  this  incision  enough  material  may  be  obtained  for 
microscopic  examination  and  for  the  preparation  of 
cultures.  Fluid  exudates  that  must  be  taken  to  the 
laboratory  should  be  collected  in  either  a  sterile  test- 
tube,  or,  better,  in  a  sterile  capillary  tube  that  is 
subsequently  sealed  at  both  ends  in  a  gas-flame. 
When  bacteriological  examination  of  the  blood  dur- 
ing life  is  required,  it  is  customary  to  obtain  the  neces- 
sary sample  of  blood  by  pricking  the  skin.  It  must 
be  remembered,  in  this  connection,  that  the  skin  usu- 
ally contains  a  number  of  species  of  bacteria  that 


1264  BACTERIOLOGY. 

ar<>  of  no  pathological  significance  and  have  nothing 
to  do  with  the  disease  from  which  the  individual 
may  be  suffering.  It  is  manifestly  essential  to  ex- 
clude these.  It  is  not  possible  to  exclude  them  cer- 
tainly and  completely  under  all  circumstances,  without 
a  more  or  less  elaborate  procedure ;  but  an  effort  to  do 
so  should  always  be  made.  As  a  rule,  the  greater  num- 
ber of  them  may  be  removed  from  the  skin  by  careful 
washing  with  warm  water  and  soap  and  a  sterile  brush, 
after  which  the  skin  should  be  rinsed  with  alcohol  and 
allowed  to  dry  spontaneously.  The  drop  of  blood  may 
then  be  obtained  from  the  skin  thus  cleaned  by  a  prick 
with  a  sharp,  sterilized  lancet.  The  presence  in  the 
cultures  of  a  staphylococcus,  growing  slowly,  with 
white  colonies,  is  a  frequent  experience,  and  does  not 
necessarily  imply  that  this  organism  bears  an  etiological 
relation  to  the  disease  from  which  the  individual  may 
be  suffering  (see  staphyloeoccus  epidermidis  (tlbuti,  page 
281). 

In  the  study  of  many  of  the  common  diseases, 
notably  the  exanthemata,  both  at  autopsy  and  during 
life,  by  the  methods  above  outlined,  the  investigation 
often  yields  negative  results,  and  yet  there  is  every 
reason  for  believing  these  diseases  to  be  dependent  for 
their  existence  upon  invasion  of  the  body  by  some  form 
or  another  of  living  micro-organisms,  capable  of  growth 
in  the  tissues  and  susceptible  of  being  transmitted  from 
individual  to  individual,  either  directly  or  indirectly. 
In  this  connection  it  is  appropriate  to  call  attention 
to  the  novel  and  important  technical  procedures  that 
have  been  employed  by  Nocard  and  Roux l  in  their 
investigations  conducted  in  the  Pasteur  Institute  at 

1  Nocard  and  Koux  :  Annales  de  1'Institiit  Pasteur,  April  25,  1898. 


STUDIES  OF  TISSUES  DURING  LIFE.          265 

Paris.  In  the  course  of  their  studies  upon  the  pleuro- 
pneumonia  of  cattle,  a  disease  in  which  all  investigators 
had  hitherto  failed  to  detect  by  either  microscopic  or 
culture-methods  any  species  of  bacteria  that  might  rea- 
sonably be  regarded  as  the  causative  agent,  they  detected 
a  group  of  bodies,  apparently  bacteria,  of  such  infinitesi- 
mally  small  dimensions  as  entirely  to  escape  detection 
by  the  usual  methods  of  examination. 

The  results  of  Nocard  and  Roux  were  obtained  both 
through  the  adoption  of  special  methods  of  cultivation 
and  the  use  of  very  high  amplifying  powers  for  micro- 
scopic examination.  The  method  of  cultivation  was  that 
suggested  in  1896  by  Metchnikoif,  Roux,  and  Salimbeni,1 
and  is  essentially  as  follows :  very  thin-walled,  small 
sacs  of  collodion  are  sterilized  by  steam,  filled  with 
bouillon,  inoculated  with  the  exudate  or  tissue  to  be 
tested,  sealed  with  sterile  collodion,  and  placed  in  the 
abdominal  cavity  of  an  animal  —  rabbit,  guinea-pig, 
chicken,  dog,  sheep,  or  calf,  as  the  case  may  require. 
The  wall  of  the  collodion  sac  is  impermeable  to  bac- 
teria or  to  leucocytes,  but  is  an  osmotic  membrane 
through  which  fluids  and  some  of  their  dissolved 
contents  readily  diffuse.  This  diffusion  supplies  the 
bacteria  within  the  sacs  with  such  matters  from  the 
living  fluids  of  the  animal  as  are  apparently  essential  to 
their  development,  while  at  the  same  time  the  bacteria 
develop  uninterruptedly,  being  protected  by  the  collodion 
membrane  from  the  antagonistic  action  of  the  fixed  and 
wandering  cells  of  the  tissues.  After  a  period  of  from  a 
few  days  to  several  months  the  animal  is  sacrificed,  and 
the  sac  removed  from  the  peritoneal  cavity  and  its  con- 

1  Metchnikoft',  Roux,  and  Salimbeni:  Annules  de  1'Institut  Pasteur, 
189(i,  p.  257. 


266  BA  CTERIOLOG  Y. 

tents  subjected  to  microscopic  examination.  This  latter 
part  of  the  work  was  unsatisfactory  when  conducted  with 
the  usual  combinations  of  lenses  employed  in  bacterio- 
logical work.  Satisfactory  examinations  could  only  be 
made  by  the  use  of  very  high  magnifying  powers,  about 
2000  diameters,  and  unusually  brilliant  illumination. 
When  conducted  under  these  conditions  the  sacs  inoc- 
ulated with  matters  from  the  pulmonary  exudates  of 
pleuro-pneumonia  were  found  to  contain  numerous  mo- 
tile points  or  dots  of  such  extremely  small  size  that  it 
was  often  impossible  to  decide  as  to  their  exact  form. 
Control-sacs  not  inoculated,  but  kept  in  the  peritoneal 
cavity  of  the  same  animal,  manifestly  under  similar  con- 
ditions, did  not  reveal  the  presence  of  the  minute  bodies. 
In  referring  at  length  to  this  investigation  it  is  not 
my  purpose  to  discuss  the  object  of  it,  but  only  to 
direct  attention  to  this  novel  technique,  which  seems 
capable  of  much  wider  application.  There  is  a  group 
of  common  maladies,  such  as  measles,  scarlet  fever, 
smallpox,  etc.,  of  the  etiology  of  which  we  know  noth- 
ing, and  on  which  it  has  hitherto  been  impossible 
to  shed  important  light  by  the  usual  bacteriological 
procedures.  Nocard  and  Roux  have  apparently  re- 
vealed to  us  a  world  of  micro-organisms  whose  existence 
has  hitherto  been  unsuspected,  and  it  is  not  unreason-  , 
able  to  suppose  that  it  is  among  this  group  that  we  are 
to  seek  for  the  causative  agents  of  many  specific  dis- 
eases whose  etiology  is  as  yet  obscure.1 

1  An  excellent   review  of  the  paper  of  Nocard  and  Roux  is  to  be 
found  in  the  Philadelphia  Medical  Journal  of  June  11,  1898. 


APPLICATION    OF  THE   METHODS    OF 
BACTERIOLOGY. 


CHAPTER    XIV. 

To  obtain  material  with  which  to  begin  work. 

EXPOSE  to  the  air  of  an  inhabited  room  a  slice  of 
freshly  steamed  potato  or  a  bit  of  slightly  moistened 
bread  upon  a  plate  for  about  one  hour.  Then  cover  it 
with  an  ordinary  water-glass,  place  it  in  a  warm  spot 
(temperature  not  to  exceed  that  of  the  human  body 
—37.5°  C.),  and  allow  it  to  remain  undisturbed.  In 
from  twenty-four  to  thirty-six  hours  there  will  be  seen 
upon  the  cut  surface  of  the  bread  or  potato  small, 
round,  oval,  or  irregularly  round  patches  which  present 
various  appearances.  These  differences  in  macroscopic 
appearance  are  due  in  some  cases  to  the  presence  or 
absence  of  color;  in  others  to  a  higher  or  lower 
degree  of  moisture ;  in  some  instances  a  patch  will  be 
glistening  and  smooth,  while  its  neighbor  may  be  dull 
and  rough  or  wrinkled ;  here  will  appear  an  island 
regularly  round  in  outline,  and  there  an  area  of 
irregular,  ragged  deposit.  All  these  gross  appear- 
ances are  of  value  in  aiding  us  to  distinguish  between 
these  colonies — for  colonies  they  are,  and  under  the 
same  conditions  the  organisms  composing  each  of  them 
will  always  produce  growth  of  exactly  the  same  ap- 
pearance. It  was  just  such  an  experiment  as  this, 

267 


268  EACTERIOLOG  Y. 

accidentally  performed,  that,  suggested  to  Koeli  a  means 
of  separating  and  isolating  in  pure  cultures  the  com- 
ponent individuals  from  mixtures  of  bacteria,  and  it 
was  from  this  observation  that  the  methods  of  cultiva- 
tion on  solid  media  were  evolved. 

If,  without  molesting  these  objects,  we  continue  the 
observations  from  day  to  day,  we  shall  notice  changes 
in  the  colonies,  due  to  the  growth  and  multiplication  of 
the  individuals  composing  them.  In  some  cases  the 
colonies  will  always  retain  their  sharply  cut,  round,  or 
oval  outline,  and  will  increase  but  little  in  size  beyond 
that  reached  after  forty-eight  to  seventy-two  hours ; 
whereas  others  will  spread  rapidly  and  quickly  overrun 
the  surface  upon  which  they  are  growing,  and,  indeed, 
grow  over  the  smaller,  less  rapidly  developing  colonies. 
In  a  number  of  instances,  if  the  observation  be  con- 
tinued long  enough,  many  of  these  rapidly  growing 
colonies  will,  after  a  time,  lose  their  lustrous  and  smooth 
or  regular  surface  and  will  show  here  and  there  eleva- 
tions, which  will  continue  to  appear  until  the  whole 
surface  becomes  conspicuously  wrinkled.  Again,  bub- 
bles may  be  seen  scattered  through  the  colonies.  These 
are  due  to  the  escape  of  gas  resulting  from  fermentation, 
which  the  organisms  bring  about  in  the  medium  upon 
which  they  are  growing.  Sometimes  peculiar  odors  due 
to  the  same  cause  will  be  noticed. 

Note  carefully  all  these .  changes  and  appearances,  as 
they  must  be  employed  subsequently  in  identifying  the 
individual  organisms  from  which  each  colony  on  the 
medium  has  developed. 

If  now  we  examine  these  colonies  upon  the  bread  or 
potato  with  a  hand-lens  of  low  magnifying  power,  we 
will  be  enabled  to  detect  differences  not  noticeable  to 
the  naked  eye.  In  a  few  cases  we  may  still  see  nothing 


MATERIAL    WITH   WHICH  TO  BEGIN   WORK.  269 

more  than  a  smooth,  non-characteristic  surface ;  while 
in  others  minute,  sometimes  regularly  arranged  tiny 
corrugations  may  be  observed.  In  one  colony  they  may 
appear  as  tolerably  regular  lines,  radiating  from  a  cen- 
tral spot ;  and  again  they  may  appear  as  concentric 
rings ;  and  if  by  the  methods  which  have  been  de- 
scribed we  obtain  from  these  colonies  their  individual 
components  in  pure  culture,  we  shall  see  that  this 
characteristic  arrangement  in  folds,  radii,  or  concentric 
rings,  or  the  production  of  color,  is  characteristic  of  the 
growth  of  the  organism  under  the  conditions  first 
observed,  and  by  a  repetition  of  those  conditions  may 
be  reproduced  at  will. 

So  much  for  the  simplest  naked-eye  experiment  that 
can  be  made  in  bacteriology,  and  which  serves  to  furnish 
the  beginner  with  material  upon  which  to  commence  his 
studies.  It  is  not  necessary  at  this  time  for  him  to  bur- 
den his  mind  with  names  for  these  organisms ;  it  is  suffi- 
cient for  him  to  recognize  that  they  are  mostly  of  differ- 
ent species,  and  that  they  possess  characteristics  which 
will  enable  him,  to  differentiate  the  one  from  the  other. 

In  order  now  for  him  to  proceed  it  is  necessary  that 
he  should  have  familiarized  himself  with  the  methods 
by  which  his  media  are  prepared  and  the  means  em- 
ployed in  sterilizing  them  and  retaining  them  sterile — 
i.  e.y  of  preventing  the  access  of  foreign  germs  from 
without — otherwise  his  efforts  to  obtain  and  retain  his 
organisms  as  pure  cultures  will  be  in  vain. 

EXPOSURE  AND  CONTACT. — Make  a  number  of  plates 
from  bits  of  silk  used  for  sutures,  after  treating  them 
as  follows  : 

Place  some  of  the  pieces  (about  5  centimetres  long) 
in  a  sterilized  test-tube,  and  sterilize  them  by  streaming 
for  one  hour  or  in  the  autoclave  for  fifteen. min- 


270  BA  CTERIOLOG  Y. 

utes  at  one  atmosphere  pressure.  At  the  end  of  the 
sterilization  remove  one  pieco  with  sterilized  forceps  and 
allow  it  to  brush  against  your  clothing,  then  make  a 
plate  from  it ;  draw  another  piece  across  a  dusty  table 
and  then  plate  it.  Suspend  three  or  four  pieces  upon  a 
sterilized  wire  hook  and  let  them  hang  for  twenty  min- 
utes free  in  the  air,  being  sure  that  they  touch  nothing 
but  the  hook;  then  plate  them  separately. 

Note  the  results. 

In  what  way  do  these  experiments  diiFer  and  how 
can  the  differences  be  explained  ? 

Expose  to  the  air  six  Petri  dishes  into  which  either 
sterilized  gelatin  or  agar-agar  has  been  poured  and 
allowed  to  solidify ;  allow  them  to  remain  exposed  for 
five,  ten,  fifteen,  twenty,  twenty-five,  and  thirty  min- 
utes in  a  room  where  no  one  is  at  work.  Treat  a  sec- 
ond set  in  the  same  way  in  a  room  where  several  persons 
are  moving  about.  Be  careful  that  nothing  touches  them, 
and  that  they  are  exposed  only  to  the  air.  Each  dish 
should  be  carefully  labelled  with  the  time  of  its  exposure. 

Do  they  present  different  results  ?  What  is  the  rea- 
son for  this  difference  ? 

Which  predominate — colonies  resulting  from  the 
growth  of  bacteria,  or  those  from  common  moulds  ? 

How  do  you  account  for  this  condition  ? 

Sprinkle  a  little  fine  dust  over  the  surface  of  a  plate 
of  sterile  gelatin  or  agar-agar ;  examine  the  dust-par- 
ticles with  the  microscope  immediately  after  depositing 
them  on  the  medium,  and  again  after  eighteen  to  twenty- 
four  hours.  What  differences  do  you  detect?  What 
information  of  sanitary  importance  does  this  give  ? 

Under  the  description  of  each  of  the  pathogenic  bacte- 
ria more  or  less  detailed  directions  will  be  found  for  the 
discovery  and  isolation  of  each  of  the  pathogenic  bacteria, 


CHAPTER    XV. 

Suppuration — Micrococcus  aureus — Micrococcus  pyogenes  and  citreus 
— Staphylococcus  epidermidis  albus — Streptococcus  pyogenes — 
Micrococcus  gonorrhoea — Micrococcus  intracellularis — Pseudomo- 
nas  seruginosa — Bacillus  of  bubonic  plague — Bacterium  pseudo- 
diphtheriticum. 

MICROCOCCUS   AUREUS    (ROSENBACH),    MIGULA,    1900. 

Synonyms:  Staphylococcus  pyogenes  aureus,  Rosenbach,  1884;  Micro- 
coccus  pyogenes  aureus,  Migula,  1895 ;  Micrococcus  pyogenes,  Leh- 
manii  and  Neumann,  1896. 

PREPARE  a  set  of  plates  of  agar-agar  from  the  pus 
of  an  acute  abscess  or  boil  that  has  been  opened  under 
antiseptic  precautions.  Care  must  be  taken  that  none 
of  the  antiseptic  used  gains  access  to  the  culture-tubes, 
otherwise  its  antiseptic  effect  may  be  operative  and  the 
development  of  the  organisms  interfered  with.  It  is 
best,  therefore,  to  take  a  drop  of  the  pus  upon  a  plati- 
num-wire loop  after  it  has  been  flowing  for  a  few  sec- 
onds ;  even  then  it  must  be  taken  from  the  mouth  of 
the  incision  and  before  it  has  run  over  the  surface  of  the 
skin.  At  the  same  time  prepare  two  or  three  cover- 
slips  from  the  pus. 

Microscopic  examination  of  these  slips  will  reveal  the 
presence  of  a  large  number  of  pus-cells,  both  multi- 
nucleated  and  with  horseshoe-shaped  nuclei,  some 
threads  of  disintegrated  and  necrotic  connective  tissue, 
and,  lying  here  and  there  throughout  the  preparation, 
small  round  bodies  which  will  sometimes  appear  singly, 
sometimes  in  pairs,  and  frequently  will  be  seen  grouped 
together  somewhat  like  clusters  of  grapes.  (See  Fig. 
54.)  They  stain  readily  and  are  commonly  located  in 
the  material  between  the  pus-cells;  very  rarely  they 
may  be  seen  in  the  protoplasmic  body  of  the  cell. 
(Compare  the  preparation  with  a  similar  one  made 

271 


272  BACTERIOLOGY. 

from  the  pus  of  gonorrhoea.  (See  Fig.  57.)  In  what 
way  do  the  two  preparations  differ,  the  one  from  the 
other?) 

FIG.  54. 


Preparation  from  pus,  showing  pus-cells,  A,  and  micrococci,  C. 

After  twenty-four  hours  in  the  incubator  the  plates 
will  be  seen  to  be  studded  here  and  there  with  yellow 
or  orange-colored  colonies,  which  are  usually  round, 
moist,  and  glistening  in  their  naked-eye  appearances. 
When  located  in  the  depths  of  the  medium  they  are 
commonly  seen  to  be  lozenge  or  whetstone  in  shape, 
while  often  they  appear  as  irregular  stars  with  blunt 
points,  and  again  as  irregularly  lobulated  dense  masses. 
In  structure  they  are  conspicuous  for  their  density. 
Under  the  low  objective  they  appear,  when  on  the  sur- 
face, as  coarsely  granular,  irregularly  round  patches, 
with  more  or  less  ragged  borders  and  a  dark  irregular 
central  mass,  which  has  somewhat  the  appearance  of 
masses  of  coarser  clumps  of  the  same  material  as  that 
composing  the  rest  of  the  colony.  Microscopically, 
these  colonies  are  composed  of  small  round  cells,  irreg- 
ularly grouped  together.  They  are  in  every  way  of 


M1CROCOCCUS  AUREUS. 

the  saint!  appearance  as  those  seen  upon  the  original 
cover-slip  preparation. 

Prepare  from  one  of  these  colonies  a  pure  stab-culture 
in  gelatin.  After  thirty-six  to  forty-eight  hours  lique- 
faction of  the  gelatin  along  the  track  of  the  needle, 
most  conspicuous  at  its  upper  end,  will  be  observed. 
As  growth  continues  the  liquefied  portion  becomes  more 
or  less  of  a  stocking-shape,  and  gradually  widens  at  its 
upper  end  into  an  irregular  funnel.  This  will  continue 
until  the  whole  of  the  gelatin  in  the  tube  eventually 
becomes  fluid.  There  can  always  be  noticed  at  the 
bottom  of  the  liquefying  portion  an  orange-colored  or 
yellow  mass  composed  of  a  number  of  the  organisms 
which  have  sunk  to  the  bottom  of  the  fluid. 

On  potato  the  growth  is  quite  luxuriant,  appearing  as 
a  brilliant,  orange-colored  layer,  somewhat  lobulated  and 
a  little  less  moist  than  when  growing  upon  agar-agar. 

It  does  not  produce  fermentation  with  gas-production. 

It  belongs  to  the  group  of  facultative  anaerobes. 

In  milk  it  rapidly  brings  about  coagulation  with  acid 
reaction. 

It  is  not  motile,  and  being  of  the  family  of  micrococci 
does  not  form  endogenous  spores.  It  possesses,  how- 
ever, a  degree  of  resistance  to  detrimental  agencies  that 
is  somewhat  greater  than  that  common  to  non-spore- 
bearing  bacteria. 

In  bouillon  it  causes  a  diffuse  clouding,  and  after  a 
time  a  yellow  or  orange-colored  sedimentation. 

This  organism  is  the  commonest  of  the  pathogenic 
bacteria  with  which  we  shall  meet.  It  is  microeoccus 
(in-reus,  and  is  the  organism  most  frequently  concerned 
in  the  production  of  acute,  circumscribed,  suppurative 
inflammations.  It  is  almost  everywhere  present,  and 

18 


274  BA  CTERIOLOG  Y. 

is  the  organism  causing  continuous  annoyance  to  the 
surgeon. 

In  studying  its  effects  upon  lower  animals  a  number 
of  points  are  to  be  remembered.  While  i-t  is  the  etio- 
logical  factor  in  the  production  of  most  of  the  snppu- 
rative  processes  in  man,  still  it  is  with  no  little  difficulty 
that  these  conditions  can  be  reproduced  in  lower  ani- 
mals. Its  subcutaneous  introduction  into  their  tissues 
does  not  always  result  in  abscess-formation,  and  when  it 
does  there  seems  to  have  been  some  coincident  interfer- 
ence with  the  circulation  and  nutrition  of  these  tissues 
which  renders  them  less  able  to  resist  its  inroads.  When 
introduced  into  the  great  serous  cavities  of  the  lower 
animals  its  presence  is  likewise  not  always  accompanied 
by  the  production  of  inflammation.  If  the  abdominal 
cavity  of  a  dog,  for  example,  be  carefully  opened  so  as 
to  make  as  slight  a  wound  as  possible,  and  no  injury  be 
done  to  the  intestines,  large  quantities  of  bouillon  cult- 
ures or  watery  suspensions  of  this  organism  may  be, 
and  repeatedly  have  been  introduced  into  the  peritoneum 
without  the  slightest  injury  to  the  animal.  On  the  con- 
trary, if  some  substance  which  acts  as  a  direct  irritant 
to  the  intestines — such,  for  example,  as  a  small  bit  of 
potato  upon  which  the  organisms  are  growing — be  at 
the  same  time  introduced,  or  the  intestines  be  mechani- 
cally injured,  so  that  there  is  a  disturbance  in  their  cir- 
culation, then  the  introduction  of  these  organisms  is 
promptly  followed  by  acute  and  fatal  peritonitis.  (Hal- 
sted.1) 

On  the  other  hand,  the  results  which  follow  their  in- 
troduction into  the  circulation  are  practically  constant. 

1  Halsted :  The  Johns  Hopkins  Hospital  Eeports.     Report  in  Sur- 
gery, No.  1,  1891,  vol.  ii.  No.  5,  pp.  301-303. 


Mic&ocuccirs  AUREUS.  275 

If  one  inject  into  the  circulation  of  the  rabbit  through 
a  vein  of  the  ear,  or  in  any  other  way,  from  0.1  to 
0.3  c.c.  of  a  bouillon  culture  or  watery  suspension  of 
a  virulent  variety  of  this  organism,  a  fatal  pyaemia 
always  follows  in  from  two  and  one-half  to  three  days. 
A  few  hours  before  death  the  animal  suffers  frequently 
from  severe  convulsions.  Now  and  then  excessive 
secretion  of  urine  is  noticed.  The  animal  may  appear 
in  moderately  good  condition  until  from  eight  to  ten 
hours  before  death.  At  the  autopsy  a  typical  picture 
presents :  the  voluntary  muscles  are  seen  to  be  marked 
here  and  there  by  yellow  spots,  which  average  the  size 
of  a  flaxseed,  and  are  of  about  the  same  shape.  They 
lie  usually  with  their  long  axis  running  longitudinally 
between  the  muscle-fibres.  As  the  abdominal  and  tho- 
racic cavities  are  opened  the  diaphragm  is  often  seen  to 
be  studded  with  them.  Frequently  the  pericardial  sac 
is  distended  with  a  clear  gelatinous  fluid,  and  almost 
constantly  the  yellow  points  are  seen  in  the  myocar- 
dium. The  kidneys  are  rarely  without  them ;  here 
they  appear  on  the  surface  as  isolated  yellow  points, 
or,  again,  are  seen  as  conglomerate  masses  of  small 
yellow  points  which  occupy,  as  a  rule,  the  area  fed 
by  a  single  vessel.  If  one  make  a  section  into  one 
of  these  yellow  points,  it  will  be  seen  to  extend  deep 
down  through  the  substance  of  the  kidney  as  a  yellow, 
wedge-shaped  mass,  the  base  of  the  wedge  being  at  the 
surface  of  the  organ. 

It  is  very  rare  that  these  abscesses — for  abscesses  the 
yellow  points  are,  as  we  shall  see  when  we  come  to  study 
them  more  closely — are  found  either  in  the  liver,  spleen, 
or  brain ;  their  usual  location  being,  as  said,  in  the  kid- 
ney, myocardium,  and  voluntary  muscles. 


276  BA  CTERIOL  OGY. 

These  minute  abscesses  contain  a  dry,  cheesy,  necrotic 
centre,  in  which  the  micrococci  are  present  in  large 
numbers,  as  may  be  seen  upon  cover-slips  prepared 
from  them.  This  organism  may  also  be  obtained  in 
pure  culture  from  the  suppurating  foci. 

Preserve  in  Miiller's  fluid  and  in  alcohol  duplicate 
bits  of  all  the  tissues  in  which  the  abscesses  are  located. 
When  these  tissues  are  hard  enough  to  cut,  sections 
should  be  made  through  the  abscess-points  and  the  his- 
tological  changes  carefully  studied. 

MICROSCOPIC  STUDY  OF  COVER-SLIPS  AND  SECTIONS. 
—In  cover-slip  preparations  this  organism  stains  readily 
with  the  ordinary  dyes.  In  tissues,  however,  it  is  best 
to  employ  some  method  by  means  of  which  contrast- 
stains  may  be  utilized,  and  the  location  and  grouping  of 
the  organisms  in  the  tissues  rendered  more  conspicuous. 
When  stained,  sections  of  tissues  containing  the  small 
abscesses  present  the  following  appearances  : 

To  the  naked  eye  will  be  seen  here  and  there  in  the 
section,  if  the  abscesses  are  very  numerous,  small,  darkly 
stained  areas  which  range  in  size  from  that  of  a  pin- 
point up  to  those  having  a  diameter  of  from  1  to  2  mm. 
These  points,  when  in  the  kidney,  may  be  round  or  oval 
in  outline ;  or  may  appear  wedge-shaped,  with  the  base 
of  the  wedge  toward  the  surface  of  the  organ.  The 
differences  in  shape  depend  frequently  upon  the  direction 
in  which  the  section  has  been  made  through  the  kidney. 
In  the  muscles  they  are  irregularly  round  or  oval. 

When  quite  small  they  appear,  in  stained  sections,  to 
the  naked  eye,  as  simple,  round  or  oval,  darkly  stained 
points ;  but  when  they  are  in  a  more  advanced  stage  a 
pale  centre  can  usually  be  made  out. 

When  magnified  they  appear  in  the  earliest  stages  as 


COVER-SLIPS  AND  SECTIONS.  277 

minute  aggregations  of  small  cells,  the  nuclei  of  which 
stain  intensely.  Almost  always  evidences  of  progress- 
ing necrosis  can  be  seen  about  the  centre  of  these  cell- 
accumulations.  The  normal  structure  of  the  cells  of 
the  tissues  is  more  or  less  destroyed ;  there  is  seen 
a  granular  condition  due  to  cell-fragmentation ;  at  dif- 
ferent points  about  the  centre  of  this  area  the  tissue 
appears  cloudy  and  the  tissue-cells  do  not  stain  read- 
ily. Round  about  and  through  this  spot  are  seen 
the  nuclei  of  pus-cells,  many  of  which  are  undergoing 
disintegration.  In  the  smallest  of  these  beginning  ab- 
scesses the  micrococci  are  to  be  seen  scattered  about 
the  centre  of  the  necrotic  tissue  ;  but  in  a  more  advanced 
stage  they  are  commonly  seen  massed  together  in  very 
large  numbers  in  the  form  commonly  referred  to  as 
emboli  of  microcoeci. 

When  the  process  is  well  advanced,  the  different  parts 
of  the  abscess  are  more  easily  detected.  They  then  pre- 
sent in  sections  somewhat  the  following  conditions :  at 
the  centre  can  be  seen  a  dense,  granular  mass  which 
stains  readily  with  the  basic  aniline  dyes,  and  when 
highly  magnified  is  found  to  be  made  up  of  micro- 
cocci.  Sometimes  the  shape  of  this  mass  of  micro- 
cocci  corresponds  to  that  of  the  capillary  in  which  the 
organisms  became  lodged  and  developed.  Immediately 
about  the  embolus  of  cocci  the  tissues  are  in  an  advanced 
stage  of  necrosis.  Their  structure  is  almost  completely 
destroyed,  although  the  destruction  is  seen  to  be  more 
advanced  in  some  of  the  elements  of  the  tissues  than  in 
others.  As  we  approach  the  periphery  of  this  faintly 
stained  necrotic  area  it  becomes  marked  here  and  there 
with  granular  bodies,  irregular  in  size  and  shape,  which 
stain  in  the  same  way  as  do  the  nuclei  of  the  pus-cells 


278  BACTERIOLOG  Y. 

and  represent  the  result  of  disintegration  going  on  in 
these  eells. 

Beyond  this  area  we  come  upon  a  dense,  deeply  stained 
zone,  consisting  of  closely  packed  pus-cells  ;  of  granular 
detritus  resulting  from  destructive  processes  acting  upon 
these  cells ;  and  of  the  normal  cellular  and  connective- 
tissue  elements  of  the  part.  Here  and  there  through 
this  zone  will  be  seen  localized  areas  of  beginning  death 
of  the  tissues.  This  zone  gradually  fades  away  into 
the  healthy  surrounding  tissues.  It  constitutes  the  so- 
called  "  abscess- wall.77 

Such  is  the  picture  presented  by  the  miliary  abscess 
when  produced  experimentally  in  the  rabbit,  and  it  cor- 
responds from  beginning  to  end  with  the  pathological 
changes  which  accompany  the  formation  of  larger  ab- 
scesses in  the  tissues  of  human  beings. 

From  these  small  abscesses  in  the  tissues  of  the 
rabbit  micrococeus  aureus  may  again  be  obtained  in  pure 
culture,  and  will  present  identically  the  same  character- 
istics that  were  possessed  by  the  culture  with  which  the 
animal  was  inoculated. 

A  characteristic  of  all  staphylococcus  abscesses,  small 
as  well  as  large,  is  centralized  death  of  tissue ;  even  in 
those  of  microscopic  dimensions  this  area  of  necrosis 
is  always  discernible  by  appropriate  methods  of  exami- 
nation. It  represents  the  very  starting-point  of  the 
destructive  process,  and  is  referable  to  the  action  upon 
the  tissues  of  poisons  elaborated  by  living  bacteria  that 
have  gained  access  to  them. 

As  a  result  of  the  studies  of  van  de  Velde,  Krauss, 
von  Lingelsheim,  Neisser  and  Wechsberg,  and  others, 
our  knowledge  of  the  poison  that  causes  the  destruction 

staphylotoxin,  as  it  is  called — has  been  greatly  ex- 


COVER-SLIPS  AND  SECTIONS.  279 

tended.  Through  the  work  of  these  investigators  we  now 
know  that  the  pathogenic  properties  of  staphylococcus 
pyogenes  auretis  are  due  to  a  definite  soluble  toxin  elab- 
orated by  it :  that  this  poison  is  produced  under  arti- 
ficial conditions  of  cultivation,  and  may  be  separated 
from  the  living  organisms  by  filtration ;  that  when 
injected  into  the  living  animal  body  its  effects  upon  the 
tissues  are  essentially  reproductions  of  those  accompany- 
ing the  growth  of  the  organism  itself;  that  when  this 
action  is  tested  upon  particular  cells,  such  as  erythro- 
cytes  and  leucocytes,  two  distinct  properties  are  exhib- 
ited, one  a  hsemolytic,  through  which  the  red  corpuscles 
of  the  blood  are  dissolved,  the  other  a  leucocidic,  through 
which  the  white  blood-corpuscles  are  destroyed ;  that 
the  hsemolytic  and  leucocidic  properties  are  distinct 
from  one  another,  and  are  due  to  the  activities  of  two 
lysins,  of  which  the  staphylotoxin  is  (in  part?)  com- 
posed, and  which  may  be  separated  from  one  another  by 
appropriate  methods  of  analysis ;  that  the  result  of  the 
treatment  of  animals  with  gradually  increasing  non-fatal 
doses  of  staphylotoxin  is  the  appearance  in  the  blood  of 
the  animals  of  antitoxic  bodies  (antilysins)  that  inhibit 
the  action  of  the  toxins  (lysins) ;  and,  finally,  that  in 
the  serum  of  certain  animals  (man  and  horse)  similar 
antilysins  in  varying  amounts  are  normally  present.1 

Petersen,  Paltchikowsky,  Proscher,  and  others  have 
recently  attempted  to  prepare  an  antistaphylococcus 
serum.  The  serum  of  patients  recovering  from  severe 
staphylococcus  infections  contains  protective  substances 

1  See  van  de  Velde :  Annales  de  1'Institut  Pasteur,  tome  x.  p.  580. 
Krauss:  Wiener  klin.  Wochenschrift,  1900,  No.  3.  Von  Lingelsheim: 
Etiologie  und  Therapie  der  Staphylokoken  Infektiou  (monograph), 
Berlin- Wien,  1900.  Neisser  and  Wechsnerg:  Zeitschrift  fur  Hygiene 
und  Infektiouskrankkeiten,  1901,  Bd.  xxxvi.  S.  299. 


280  BACTERIOLOGY. 

which  serve  to  protect  rabbits  from  twice  the  fatal  dose 
of  a  staphylococcus  culture.  Similarly  the  serum  of 
immunized  rabbits  and  goats,  as  shown  by  the  exper- 
iments of  Petersen,  possesses  about  the  same  degree  of 
protective  powers.  No  antitoxic  power  could  be  dem- 
onstrated in  the  serum  of  the  treated  animals.  The 
extremely  limited  degree  of  the  protective  power  of  the 
antistaphylococcus  serums  prepared  thus  far  makes  it 
impossible  to  employ  them  for  curative  purposes  in 
human  beings,  as  Petersen  calculated  that  an  adult 
would  require  from  350  to  700  c.c.  of  the  serum  at  a  sin- 
gle dose,  as  judged  by  its  effects  on  the  lower  animals. 

OTHER   COMMON   PYOGENIC   ORGANISMS. 

MICROCOCCUS  PYOGENES  (Rosenbach),  Migula,  1900.  Synonyms: 
Staphylococcus  pyogenes  albus,  Rosenbach,  1884 ;  Micrococcus  pyogenes 
albus,  Lehmann  and  Neumann,  1896. 

MICROCOCCUS  CITREUS  (Passet),  Migula,  1900.  Synonym:  Staphylo- 
coccus pyogenes  citreus,  Passet,  1885. 

The  pus  of  an  acute  abscess  in  the  human  being 
may  sometimes  contain  organisms  other  than  micro- 
coccus  aureus.  Micrococcus  pyogenes  and  citreus  may  be 
found.  The  colonies  of  the  former  are  white,  those  of 
the  latter  are  lemon  color.  With  these  exceptions  they 
are  in  all  essential  cultural  peculiarities  similar  to  micro- 
coccus  aureus.  As  a  rule,  they  are  not  virulent  for 
animals,  and  when  they  do  possess  pathogenic  proper- 
ties, it  is  in  a  much  lower  degree  than  is  commonly  the 
case  with  the  golden  staphylococcus.  Streptococcus 
pyogenes  is  also  sometimes  present.  The  commonest 
of  the  pyogenic  organisms,  however,  is  that  just  de- 
scribed, viz.,  micrococcus  aureus.  An  organism  that  is 
almost  universally  present  in  the  skin,  and  is  often  con- 
cerned in  producing  mild  forms  of  inflammation,  is 


OTHER   COMMON  PYOGENIC  ORGANISMS.      281 

staphylococcus  epidermidis  albus  (Welch),  an  organism 
that  may  readily  be  confused  with  micrococcus  pyogenes. 
It  is  distinguished  from  the  latter  by  the  slowness  with 
which  it  liquefies  gelatin  and  by  the  comparative  absence 
of  pathogenic  properties  when  injected  into  the  circula- 
tion of  rabbits.  Welch  regards  this  organism  as  a 
variety  of  micrococcus  pyogenes. 


Streptococcus  pyogenes  in  pus. 

STREPTOCOCCUS  PYOGENES  (ROSENBACH),  MIGULA, 
1900. 

Synonyms:  Streptococcus,  Billroth,  1874;   Streptococcus  pyogenes, 
Rosenbach,  1884. 

From  a  spreading  phlegmonous  inflammation  prepare 
cover-slips  and  cultures.  What  is  the  predominating 
organism  ?  Does  it  appear  in  the  form  of  irregular  clus- 
ters like  those  of  grapes,  or  have  its  individuals  a  definite, 
regular  arrangement  ?  (See  Fig.  55.)  Are  its  colonies 
like  those  of  micrococcus  aureusf 

Isolate  this  organism  in  pure  cultures.     In  these  cul- 


282  BACTERIOLOGY. 

tures  it  will  be  found  on  microscopic;  examination  to 
present  an  arrangement  somewhat  like  a  chain  of  beads. 
(Fig.  66.) 

Determine  its  peculiarities  and  describe  them  accu- 
rately. They  should  be  as  follows : 

Upon  microscopic  examination  a  micrococcns  should 
be  found,  but  differing  in  its  arrangement  from  the 
staphylococci  just  described.  The  single  cells  are  not 
scattered  irregularly  or  arranged  in  clumps  similar  to 
bunches  of  grapes,  but  are  joined  together  in  chains  like 
strands  of  beads.  These  strands  are  sometimes  regular 
in  the  arrangement  and  size  of  the  individual  cells  com- 

FIG.  56. 


; 


Streptococcus  pyogenes. 

posing  them,  but  more  commonly  certain  irregular  groups 
may  be  seen  in  them.  Here  they  appear  as  if  two  or 
three  cells  had  fused  together  to  form  a  link,  so  to  speak, 
in  the  chain,  that  is  somewhat  longer  than  the  remaining 
links  ;  again,  portions  of  the  chain  may  be  thinner  than  the 
rest,  or  it  may  appear  broken  or  ragged.  Commonly  the 
individuals  comprising  this  chain  of  cocci  are  not  round, 
but  appear  flattened  on  the  sides  adjacent  to  one  another. 
The  chains  are  sometimes  short,  consisting  of  four  to  six 
cells ;  or,  again,  they  may  be  much  longer,  and  extend  from 
a  half  to  two-thirds  across  the  field  of  the  microscope. 

Under  artificial  conditions  it  sometimes  grows  well, 
and  can  be  cultivated  through  many  generations,  while 


STREPTOCOCCUS  PYOGENES.  283 

at  other  times  it  rapidly  loses  its  vitality.  When  isolated 
from  the  diseased  area  upon  artificial  media  it  seems  to 
retain  its  vitality  for  a  longer  period  if  replanted  upon 
fresh  media  every  day  or  two  for  a  time ;  but  if  the 
first  generation  is  transplanted  and  is  allowed  to  re- 
main upon  the  original  medium,  it  is  not  uncommon 
to  find  the  organism  incapable  of  further  cultivation 
after  a  week  or  ten  days. 

Under  no  conditions  is  the  growth  of  this  organism 
very  luxuriant. 

On  gelatin  plates  its  colonies  appear  after  forty-eight 
to  seventy-two  hours  as  very  small,  flat,  round  points  of 
a  bluish-white  or  opalescent  appearance.  They  do  not 
cause  liquefaction  of  the  gelatin,  and  in  size  they  rarely 
exceed  0.6-0.8  mm.  in  diameter.  Under  low  magnify- 
ing power  they  have  a  brownish  or  yellowish  tinge  by 
transmitted  light,  and  are  finely  granular.  As  the  col- 
onies become  older  their  regular  border  may  become 
slightly  irregular  or  notched. 

In  stab-cultures  in  gelatin  they  grow  along  the  entire 
needle-track  as  a  finely  granular  line,  the  granules  rep- 
resenting minute  colonies  of  the  organism.  On  the 
surface  the  growth  does  not  usually  extend  beyond  the 
point  of  puncture. 

On  agar-agar  plates  the  colonies  appear  as  minute 
pearly  points,  which  when  slightly  magnified  are  seen 
to  be  finely  granular,  of  a  light-brownish  color,  and 
regular  at  their  margins. 

When  smeared  upon  the  surface  of  agar-agar  or  gel- 
atin slants  the  growth  that  results  is  a  thin,  pearly, 
finely  granular  layer,  consisting  of  minute  colonies 
growing  closely  side  by  side.  Its  most  luxuriant 
growth  is  seen  on  glycerin-agar-agar  at  the  tempera- 


284  BA  G  VERIO  L  OG  Y. 

ture  of  the  body  (37.5°  C.),  and  its  least  on  gelatin  at 
from  18°  to  20°  C. 

On  blood-serum  its  colonies  present  little  that  is  char- 
acteristic ;  they  appear  as  small,  moist,  whitish  points, 
from  0.6  to  0.8  mm.  in  diameter,  that  are  slightly  ele- 
vated above  the  surface  of  the  serum.  They  do  not 
coalesce  to  form  a  layer  over  the  surface,  but  remain  as 
isolated  colonies. 

On  potato  no  visible  development  appears,  but  after 
a  short  time  (thirty-six  to  seventy-two  hours)  there  is 
a  slight  increase  of  moisture  about  the  point  of  inocula- 
tion, and  microscopic  examination  shows  that  multiplica- 
tion of  the  organisms  placed  at  this  point  has  occurred. 

In  milk  its  conduct  is  not  always  the  same,  some  cult- 
ures causing  a  separation  of  the  milk  into  a  firm  clot 
and  colorless  whey,  while  others  do  not  produce  this 
coagulation.  The  latter,  when  cultivated  in  milk  of  a 
neutral  or  slightly  alkaline  reaction,  to  which  a  few 
drops  of  litmus  tincture  have  been  added,  produce,  as  a 
rule,  only  a  very  faint  pink  color  after  twenty-four  hours 
at  37.5°  C. 

In  bouillon  it  grows  as  tangled  masses  or  clumps, 
which  upon  microscopic  examination  are  seen  to  consist 
of  long  chains  of  cocci  twisted  or  matted  together. 

It  grows  best  at  the  temperature  of  the  body  (37.5° 
C.),  and  develops,  but  less  rapidly,  at  the  ordinary  room- 
temperature.  When  virulent  this  property  is  said  by 
Petruschky  to  be  preserved  by  retaining  the  cultures  in 
the  ice-chest  after  they  have  been  growing  on  gelatin 
for  two  days  at  22°  C. 

Its  virulence  may  often  be  increased  by  passing  it 
through  a  series  of  susceptible  animals. 

It  is  a  facultative  anaerobe. 


STREPTOCOCCUS  PYOGENES,  285 

It  stains  with  the  ordinary  aniline  dyes,  and  is  not 
decolorized  when  subjected  to  Gram's  method. 

It  is  not  motile,  and,  being  a  micrococcus,  does  not 
form  endogenous  spores.  Under  artificial  conditions 
we  have  no '  reason  to  believe  that  it  enters  a  stage  in 
which  its  resistance  to  detrimental  agencies  is  increased. 
In  the  tissues  of  the  body,  however,  it  appears  to  pos- 
sess marked  vitality,  for  it  is  not  rare  to  observe 
recurrences  of  inflammatory  conditions  due  to  this 
organism,  often  at  a  relatively  long  time  after  the 
primary  site  of  infection  has  healed. 

Streptococcus  pyogenes  is  the  organism  most  commonly 
found  in  rapidly  spreading  suppurations,  while  micrococ- 
cus aureus  is  most  frequently  found  in  circumscribed 
abscess  formations ;  they  may  also  be  found  together. 

The  results  of  its  inoculation  into  the  tissues  of 
lower  animals  are  described  by  Rosenbach  and  Passet 
as  protracted,  progressive,  erysipelatoid  inflammations ; 
and  Fehleisen,  who  described  a  streptococcus  in  erysip- 
elas that  is  in  all  probability  identical  with  the  strepto- 
coccus pyogenes  under  consideration,  stated  that  it  pro- 
duced in  the  tissues  of  rabbits  (the  base  of  the  ear) 
a  sharply  defined,  migratory  reddening  without  pus- 
formation.  The  writer  has  encountered  a  culture  of 
this  organism  that  possessed  the  property  of  inducing 
erysipelas  when  introduced  into  the  skin  of  the  ear,  and 
disseminated  abscess-formation  when  injected  into  the 
circulation  of  rabbits.  This  observation  has  an  im- 
portant bearing  upon  the  question  concerning  the  iden- 
tity of  streptococci  found  in  various  inflammatory  con- 
ditions, such,  for  instance,  as  the  spreading  erysipelatoid 
manifestations  on  the  one  hand,  and  the  circumscribed 
abscess-formations  on  the  other. 


280  BACTERIOLOGY. 

The  results  that  follow  upon  the  inoculation  of  ani- 
mals with  cultures  of  streptococci  obtained  from  various 
inflammatory  lesions  are,  as  a  rule,  inconstant.  At 
times  cultures  will  be  encountered  that  are  apparently 
without  virulence,  no  matter  how  tested ;  while  again 
cultures  from  other  sources  exhibit  the  most  marked 
pathogenic  properties,  even  when  employed  in  almost 
infinitesimal  quantities.  Between  these  extremes  every 
gradation  may  be  expected.  The  virulence  of  a  culture 
is  not  necessarily  proportional  to  the  intensity  of  the 
pathological  process  from  which  it  is  derived. 

There  is  never  any  certainty  of  faithfully  repro- 
ducing, by  inoculation  into  susceptible  animals,  the 
pathological  lesion  from  which  a  culture  of  the  organ- 
ism may  have  been  obtained.  The  introduction  into  a 
susceptible  animal  of  a  culture  derived  from  either  a 
spreading  phlegmon  or  an  erysipelatous  inflammation 
may  result  in  erysipelas,  general  septicaBmia,  local  ab- 
scess-formation, or,  as  said,  may  have  no  effect  at  all. 
Cultures  may  be  encountered  that  are  pathogenic  for 
one  susceptible  species  of  animals  and  not  for  another. 

Under  the  ordinary  conditions  of  artificial  cultiva- 
tion fully  virulent  varieties  of  streptococcus  pyogencs 
usually  lose  their  virulence  after  a  short  time. 
Petruschky1  preserves  this  property  by  cultivation 
upon  nutrient  gelatin  for  two  days  at  22°  C.,  keeping 
the  cultures  after  this  time  in  the  refrigerator,  and 
transplanting  upon  fresh  gelatin  every  five  or  six  days. 
Marmorek2  finds  the  virulence  preserved  by  growing 
the  organism  in  a  mixture  of  2  parts  of  horse  or 

1  Petruschky :  Centralblatt  fur  Bakteriologie  und  Parasitenkunde, 
1895,  Abth.  i.  Bd.  xvii. 

2  Marmorek :  Annales  de  1'Institut  Pasteur,  1895. 


ANT  f STREPTOCOCCUS  SERUM.  287 

human  blood-serum  and  1  part  of  nutrient  bouillon,  or 
of  1  part  of  ascites-fluid  and  2  parts  of  bouillon. 

Certain  authors  are  of  the  opinion  that  a  relation 
exists  between  virulence  and  the  length  of  the  chains 
formed  by  streptococci  when  growing  in  fluid  media. 
It  is  held  that  those  forming  the  long  chains,  strepto- 
coccus longus,  are  the  only  ones  concerned  in  animal 
pathology,  and  hence  the  only  ones  by  which  patho- 
genic powers  may  be  exhibited ;  while  those  form- 
ing the  short  chains,  streptococcus  brevis,  are  not,  as  a 
rule,  pathogenic,  and  may  often  be  readily  differentiated 
from  the  other  variety  by  more  or  less  gross  cultural 
characteristics,  such  as  slow  liquefaction  of  gelatin, 
visible  growth  on  potato,  etc.1 

ANTISTREPTOCOCCUS  SERUM. — For  a  time  a  great 
deal  of  interest  was  created  by  the  announcement  of 
Marmorek  that  he  had  succeeded  in  inducing  in  ani- 
mals a  state  of  immunity  from  streptococcus  infection, 
and  that  the  blood-serum  of  such  animals  when  injected 
into  other  susceptible  animals  and  human  beings  pos- 
sessed not  only  the  property  of  rendering  them  insus- 
ceptible to  this  particular  form  of  infection,  but  even 
exhibited  curative  powers  in  cases  already  infected. 
This  serum  was  obtained  from  horses  or  asses  that  had 
been  rendered  immune  by  the  gradual  introduction  into 
their  tissues  of  increasing  amounts  of  virulent  strepto- 
cocci. 

A  great  deal  of  experimental  work  has  been  done 
during  the  past  decade  on  the  perfection  of  an  antistrep- 
tococcus  serum.  The  views  of  the  different  experi- 

l  V.  Lingelsheim :  Zeitschrift  fur  Hygiene,  1891,  Band  x.,  and  1892, 
Band  xii.  Behring:  Centralblatt  fiir  Bakteriologie  und  Parasiten- 
kunde,  1892,  Band  xii. 


288  BACTERIOLOGY. 

mentors  differ  materially  on  certain  fundamental  points. 
Some  regard  the  streptococci  encountered  in  different 
diseases  as  possessing  specific  relations  to  such  diseases  ; 
as,  for  instance,  the  streptococcus  found  in  cases  of 
scarlet  fever  is  believed  by  Moser  and  others  to  be  specific; 
for  that  disease,  and  consequently  the  antistreptococcus 
serum  obtained  by  immunization  with  such  an  organism 
is  believed  to  possess  far  less  curative  properties  against 
other  streptococcus  infections.  If  this  idea  should 
prove  correct  then  it  will  be  necessary  to  obtain  serum 
from  animals  that  have  been  simultaneously  immun- 
ized with  a  number  of  different  streptococci  derived 
from  various  disease  conditions — a  so-called  polyvalent 
serum. 

Other  experimenters  believe  that  the  frequent  passage 
of  a  culture  of  streptococcus  through  the  lower  animals 
renders  it  less  virulent,  or  at  least  alters  its  virulence 
for  human  beings,  and  that  the  serum  obtained  through 
the  immunization  of  animals  with  such  cultures  is  less 
efficacious  than  when  the  original  virulence  of  the 
organisms  is  maintained  by  cultivation  on  suitable 
media. 

Though  all  experimental  evidence  contraindicates  the 
production  of  soluble  toxins  in  large  amounts  by  the 
streptococcus  when  grown  in  artificial  media,  Marmorek 
still  believes  that  by  special  methods  of  cultivation  the 
toxin-forming  powers  can  be  augmented,  and  that  the 
immunization  of  animals  with  such  cultures  serves  a  use- 
ful purpose  in  giving  the  serum  of  the  treated  animal  a 
more  definite  antitoxic  power. 

Aronson  prepares  his  antistreptococcus  serum  by 
immunizing  horses  with  streptococcus  cultures  that  have 
been  rendered  highly  virulent  by  repeated  passage 


ANTISTREPTOCOCCUS  SERUM.  289 

through  animals.  By  this  means  he  secures  a  20-fold 
normal  serum,  a  "  normal "  serum  being  one  of  which 
0.01  c.c.  protects  a  mouse  from  100  times  the  lethal  dose 
of  highly  virulent  streptococci.  Besides  this  the  horses 
are  subsequently  immunized  with  streptococcus  cultures 
derived  from  severe  cases  of  infection  in  human  beings 
without  passage  through  animals,  and  in  this  way  he 
believes  it  possible  to  overcome  the  objections  of  those 
who  regard  the  passage  through  animals  as  useless. 

Baginsky,  Louis  Fischer,  Charlton,  and  others  report 
having  obtained  favorable  results  in  the  treatment  of 
cases  of  scarlet  fever  complicated  with  severe  streptococ- 
cus infection.  After  several  doses  of  20  c.c.  of  the  serum 
the  fever  declined  steadily  and  continuously,  with  the 
rapid  disappearance  of  necrotic  membranes  in  the  throat, 
and  subsidence  of  the  swelling  of  the  glands  of  the 
neck. 

Foulerton l  employed  the  antistreptococcus  serum  in 
the  treatment  of  cases  of  puerperal  fever.  The  serum 
employed  was  a  polyvalent  one  derived  from  a  horse 
immunized  with  five  strains  of  streptococcus.  He 
states  that  apart  from  the  failure  of  the  serum  treat- 
ment in  puerperal  fever  arising  from  the  uncertainty  as 
to  the  particular  strain  of  streptococcus  which  is  pres- 
ent, the  question  of  the  dose  of  the  serum  to  be  employed 
is  of  considerable  importance.  He  advises  to  com- 
mence treatment  with  an  injection  of  at  least  20  c.c., 
and  if  necessary  this  is  repeated  every  twenty-four 
hours.  If  no  improvement  results  from  two  doses  of 
20  c.c.  each,  administered  within  twelve  hours,  it  is 
useless  to  persist  in  administering  it.  Large  doses  are 
necessary  for  success. 

1  Foulertou  :  The  Lancet,  Dec.  31,  1904, 
19 


290  #.4  CTERIOLOG  Y. 

Walker1  finds  that  an  injection  of  antistreptococens 
serum  in  cases  of  pure  streptococcus  infection  has  been 
followed  by  strikingly  beneficial  results.  He  believes 
the  variability  in  the  results  of  the  serum  treatment 
to  be  due  to  a  specific  affinity  of  a  serum  for  the  par- 
ticular strain  of  streptococci  used  in  producing  it,  He 
states  that  more  uniform  results  are  likely  to  be  obtained 
from  a  polyvalent  serum ;  from  the  prompt  injection  of 
serum  at  the  commencement  instead  of  near  the  close  of  a 
severe  infection  ;  and  from  the  use  of  recently  prepared 
serum.  He  also  advises  the  administration  of  the  serum 
for  some  days  after  the  general  symptoms  have  disap- 
peared, in  order  to  avoid  a  recrudescence.  The  question 
of  dose  must  be  judged  by  the  nature  of  each  case  and 
the  effect  obtained  by  the  injection,  but  it  is  important 
to  know  that  large  doses  spread  over  several  days  have 
been  used  without  ill  effect.  The  most  rational  method 
would  seem  to  be  that  of  a  large  injection  (from  20  to 
25  c.c.)  on  the  first  occasion,  followed  by  smaller  doses 
as  the  case  may  require. 

NOTE. — If  the  opportunity  presents,  obtain  cultures 
from  a  case  of  erysipelas.  Compare  the  organism 
thus  obtained  with  streptococcus  pyogenes.  Inocu- 
late rabbits  both  subcutaneously  and  into  the  circula- 
tion with  about  0.2  c.c.  of  pure  cultures  of  these  organ- 
isms in  bouillon.  Do  the  results  correspond,  and  do  they 
in  any  way  suggest  the  results  obtained  with  staphylo- 
coccus  pyogenes  aureus  when  introduced  into  animals  in 
the  same  way  ?  Do  these  streptococci  flourish  readily 
on  ordinary  media? 

i  Walker :  The  Lancet,  Dec.  31,  1904. 


LESS  COMMON  P  YOG  EN  1C  ORGANISMS.        291 


THE    LESS   COMMON    PYOGENIC    ORGANISMS. 

The  organisms  that  have  just  been  described  are 
commonly  known  as  the  "  pyogenic  cocci "  of  Ogston, 
Rosenbach,  and  Passet,  and  up  to  as  late  as  1885  were 
believed  to  be  the  specific  factors  concerned  in  the  pro- 
duction of  suppurative  inflammations.  Since  that  time, 
however,  there  has  been  considerable  modification  of 
this  view,  and  while  they  are  still  known  to  be  the 
most  common  causes  of  suppuration,  they  are  also 
known  not  to  be  the  only  causes  of  this  process. 

With  the  more  general  application  of  bacteriological 
methods  to  the  study  of  the  manifold  conditions  coming 
under  the  eye  of  the  physician,  the  surgeon,  and  the 
pathologist,  observations  are  constantly  being  made 
that  do  not  accord  with  the  earlier  ideas  upon  the 
specific  relation  of  the  pyogenic  cocci  to  all  forms  of 
suppuration.  There  is  an  abundance  of  evidence  to 
justify  the  opinion  that  a  number  of  organisms  not 
commonly  classed  as  pyogenic  may,  under  certain  cir- 
cumstances, assume  this  property.  For  example  : 

The  bacillus  of  typhoid  fever  has  been  found  in  pure 
culture  in  osteomyelitis  of  the  ribs,  in  acute  purulent 
otitis  media,  in  abscess  of  the  soft  parts,  in  the  pus 
of  empyema,  and  in  localized  fibro-peritonitis,  either 
during  its  course  or  as  a  sequel  of  typhoid  fever. 

Bacillus  coll  has  been  found  in  pure  culture  in  acute 
peritonitis,  in  liver-abscess,  in  purulent  inflammation 
of  the  gall-bladder  and  ducts,  and  in  appendicitis ;  and 
Welch  l  has  found  it  in  pure  culture  in  fifteen  different 
inflammatory  conditions. 

1  Welch  :  "  Conditions  Underlying  the  Infection  of  Wounds,"  Arneru 
can  Journal  of  the  Medical  Sciences,  November,  1891. 


292  BACTERIOLOGY. 


lanceolatus  (pneumococcus)  has  been  found 
alone  in  abscess  of  the  soft  parts,  in  purulent  infiltra- 
tion of  the  tissues  about  a  fracture,  in  purulent  cerebro- 
spinal  meningitis,  in  suppurative  synovitis,  in  acute 
pericarditis,  and  in  acute  inflammation  of  the  middle 
ear. 

Organisms  of  the  bacterium  pseudodiphtheriticum 
group  are  frequently  encountered  in  large  numbers  in 
the  pus  of  superficial  wounds,  and  especially  in  ulcera- 
tions  of  the  skin  and  mucous  membranes. 

Moreover,  many  of  the  less  common  organisms  have 
been  detected  in  pure  cultures  in  inflammatory  condi- 
tions with  which  they  were  not  previously  thought  to 
be  concerned,  and  to  which  they  are  not  usually  related 
etiologically. 

In  consideration  of  such  evidence  as  this  it  is  plain 
that  we  can  no  longer  adhere  rigidly  to  the  opinions 
formerly  held  upon  the  etiology  of  suppuration,  but 
must  subject  them  to  modifications  in  conformity  with 
this  newer  evidence.  We  now  know  that  there  exist 
bacteria  other  than  the  "  pyogenic  cocci/7  which,  though 
not  normally  pyogenic,  may  give  rise  to  tissue-changes 
indistinguishable  from  those  produced  by  the  ordinary 
pus-organisms.1 

MICROCOCCUS    GONORRHCE^E    (NEISSER),  1879. 
Synonym  :  Gonocoecus  Neisser,  Bumm,  1887. 

One  observes  upon  microscopic  examination  of  cover- 
slips  prepared  from  the  pus  of  acute  gonorrhoea  that 
many  of  the  pus-cells  contain  within  their  protoplasm 

1  Fora  more  detailed  discussion  of  the  subject,  see  "The  Factors  Con- 
cerned in  the  Production  of  Suppuration,"  International  Medical 
Magazine,  Philadelphia,  May,  1892. 


MICROCOCCUS  GONORRHCE^.  293 

numerous  small,  stained  bodies  that  are  usually  arranged 
in  pairs.  Occasionally  a  cell  is  seen  that  contains  onlv 
one  or  two  pairs  of  such  bodies ;  again,  a  cell  will  be 
encountered  that  is  packed  with  them.  Occasionally 
masses  of  these  small  bodies  will  be  seen  lying  free  in 
the  pus.  (See  Fig.  57.)  The  majority  of  the  pus-cells 
do  not  contain  them. 

These  small,  round,  or  oval  bodies  are  the  so-called 
"gonococci"  discovered  by  Neisser,  and  more  fully 
studied  subsequently  by  Bumm,  to  whom  we  are  in- 
debted for  much  of  our  knowledge  concerning  them. 

As  the  name  implies,  this  organism  is  a  micrococcus, 

FIG.  57. 

- 

, 


6 


Pus  of  gonorrhoea,  showing  diplococci  in  the  bodies  of  the  pus-cells. 

and  as  it  is  commonly  arranged  in  pairs  (flattened  at 
the  surfaces  in  juxtaposition)  it  is  often  designated  as 
diplococcus  of  gonorrhoea.  It  is  always  to  be  found  in 
gonorrhoeal  pus,  and  often  persists  in  the  urethral  dis- 
charges and  secretions  far  into  the  stage  of  conva- 
lescence. It  is  not  present  in  inflammatory  conditions 
other  than  those  of  gonorrhoeal  origin. 

It  is  easily  detected  microscopically  in  the  secretions 


294  BACTERIOLOGY. 

of  acute  gonorrhoea.  In  secondary  lesions  and  in  very 
old,  chronic  cases  it  is  difficult  of  detection  and  fre- 
quently eludes  all  efforts  to  find  it.  It  is  stained  by  the 
ordinary  methods,  but  perhaps  most  satisfactorily  with 
the  alkaline  solution  of  methylene-blue.  Most  impor- 
tant as  a  differential  test  is  its  failure  to  stain  by  the 
method  of  Gram.  (How  does  this  compare  with  the 
behavior  of  the  other  pyogenic  cocci  when  treated  in 
the  same  way  ?) 

It  does  not  grow  upon  ordinary  nutrient  media, 
and  has  only  been  isolated  in  culture  through  the  em- 
ployment of  special  methods.  Its  growth  under  arti- 
ficial conditions  seems  to  depend  upon  some  partic- 
ular nutrient  substance  that  is  supplied  by  blood  or 
blood-serum,  and  in  all  the  media  that  have  been  suc- 
cessfully used  for  its  cultivation  this  substance  is 
apparently  an  essential  constituent.  By  many  investi- 
gators it  is  thought  that  only  human  blood  possesses 
this  important  ingredient,  though  this  opinion  is  not 
universal.1 

It  was  first  isolated  in  culture  by  Bumm,  who  used 
for  this  purpose  coagulated  human  blood-serum  ob- 
tained from  the  placenta. 

Wertheim  improved  the  method  of  Bumm  by  using 
a  mixture  of  equal  parts  of  sterile  human  blood-serum 
and  ordinary  sterilized  nutrient  agar-agar,  the  latter 
having  been  liquefied  and  kept  at  50°  C.  until  after 
the  mixture  was  made,  when  it  was  allowed  to  cool  and 
solidify. 

Other  investigators  have  substituted  for  human  blood- 

1  An  instructive  article  on  this  subject  is  that  by  Foulerton :  "  On 
Micrococcus  Gonorrhoeas  and  Gonorrhosal  Infection,"  Transactions  of 
the  British  Institute  of  Preventive  Medicine,  1897,  series  i, 


MICROCOCCUS  GONOERHCE^}.  295 

serum  certain  pathological  fluids  from  the  human  body, 
such  as  ascites-fluid,  fluid  from  ovarian  cysts,  and  serous 
effusions  from  the  pleura  and  from  the  joint-cavities. 

The  method  used  by  Pfeiffer  for  the  cultivation  of 
bacterium  influenzse  is  also  said  to  have  been  success- 
fully employed.  Abel  recommends  a  needle-prick  in 
the  finger  as  a  most  convenient  source  from  which  to 
obtain  the  necessary  amount  of  human  blood  that  is  to 
be  smeared  over  the  surface  of  the  slanting  agar-agar 
when  Pfeiffer's  method  is  employed. 

Wright's  modification  of  Steinschneider's  method  has 
given  such  satisfactory  results  in  his  hands  that  it  will 
be  given  here  somewhat  in  detail.  The  medium  con- 
sists of  a  mixture  of  urine,  blood-serum  (human  or 
bovine,  either  serving  the  purpose),  and  nutrient  agar- 
agar.  The  urine  and  blood-serum  are  collected  with- 
out special  aseptic  precautions,  and  subsequently  freed 
from  bacteria  by  filtration  through  unglazed  porcelain. 
Frequently  this  is  the  tedious  part  of  the  process,  as 
the  serum  and  urine  pass  very  slowly  through  the 
porcelain  filters  generally  employed  in  laboratories. 
Wright  recommends  a  filtering  cylinder  manufactured 
by  the  Boston  Filter  Company  as  an  apparatus  that  not 
only  gives  a  sterile  filtrate,  but  also  permits  of  very 
rapid  passage  of  the  fluid. 

The  details  of  the  method  as  given  by  Wright  are  as 
follows :  "  A  litre  of  nutrient  agar  is  prepared  in  the 
usual  manner,  and  after  filtration  it  is  evaporated  to 
about  600  c.c.  This  concentration  is  desirable,  so  that 
after  dilution  with  the  urine  and  serum  the  medium 
may  be  sufficiently  firm.  This  concentrated  agar  is  then 
run  into  test-tubes  and  the  whole  sterilized  by  steam  on 
three  successive  days.  The  quantity  of  agar  placed  in 


296  BACTERIOLOGY. 

each  tube  is  smaller  than  is  usual ;  this  is  in  order  to 
allow  for  the  subsequent  addition  of  the  urine  and 
serum. 

"  The  blood-serum,  which  need  not  be  free  from  cor- 
puscles, is  first  passed  through  white  sand,  which  is 
supported  in  a  funnel  by  filter-paper,  in  order  to  re- 
move as  far  as  is  possible  any  particles  in  suspension, 
and  is  then  mixed  with  half  its  volume  of  fresh  urine. 
The  mixture  of  urine  and  blood-serum  is  next  filtered 
by  suction  through  an  unglazed  porcelain  cylinder  into 
a  receiving-flask,  such  as  chemists  use  for  similar  pur- 
poses, by  means  of  a  water-vacuum  pump.  This  frees 
the  mixture  from  bacteria. 

"  The  usual  precautions  are,  of  course,  taken  to  pre- 
vent the  contamination  of  the  filtrate,  such  as  the  pre- 
vious sterilization  by  steam  of  the  cylinder  and  receiv- 
ing-flask, besides  others  which  will  occur  to  any  bacteri- 
ologist. 

"  To  the  agar  in  each  test-tube,  which  is  fluid  and  of 
a  temperature  of  about  40°  C.,  there  is  added  about 
one-third  to  one-half  its  volume  of  the  filtered  mixture 
of  urine  and  blood-serum.  This  is  conveniently  accom- 
plished by  pouring  the  mixture  from  the  receiving-flask 
through  the  lateral  tube,  inserted  near  its  neck  directly 
into  the  tubes.  The  preliminary  melting  of  the  agar 
is  best  eifected  in  the  steam  sterilizer,  in  order  that  any 
organisms  which  have  found  lodgement  in  the  cotton 
plugs  of  the  tubes  may  be  destroyed.  When  the  agar 
is  melted  it  is  cooled  and  kept  fluid  by  placing  the 
tubes  in  a  water-bath  at  40°  C.  Each  tube,  after  the 
addition  of  the  urine  and  serum  to  the  fluid  agar,  is 
quickly  shaken  to  insure  a  uniform  mixture,  and  is 
then  placed  in  an  inclined  position  to  allow  the  agar  to 


MICROCOCCUS  GONORRHCE&.  297 

solidify  with  a  slanting  surface.  When  the  medium 
in  the  tubes  has  solidified  the  tubes  are  placed  in  the 
incubator  for  about  twenty-four  hours  to  test  for  con- 
taminations, after  which  they  are  ready  for  use." 

The  successive  dilutions  are  now  to  be  made  upon 
the  slanting  surface  of  this,  mixture,  as  the  mass  in  the 
tubes  cannot  be  redissolved  without  exposure  to  a  de- 
gree of  heat  that  apparently  interferes  with  the  nutri- 
tive value  of  the  serum  contained  in  the  medium. 

When  inoculated  with  gonorrhoeal  pus,  by  smearing 
a  loopful  over  the  surface,  the  tubes  are  to  be  kept  at 
from  37°  to  38°  C.  The  organism  does  not  develop 
properly  at  a  temperature  below  this  point. 

After  twenty-four  hours  the  colonies  of  the  gono- 
coccus  appear  on  the  surface  of  the  medium,  accord- 
ing to  Wright,  as  very  tiny,  grayish,  semi-translucent 
points.  After  forty-eight  hours  they  may  be  about 
1  millimetre  or  so  in  diameter,  slightly  elevated,  with 
a  rounded  outline,  grayish  in  color,  and  semi-translu- 
cent by  transmitted  light.  By  reflected  light  their  sur- 
face has  the  appearance  of  frosted  glass.  Later,  if  few 
in  number,  so  that  their  growth  is  unimpeded,  the  colo- 
nies may  attain  a  diameter  of  2  millimetres  or  more,  be- 
come thicker  and  denser,  with  a  faintly  brownish  tinge 
about  their  centres,  and  a  slightly  irregular  outline. 

Under  a  low  power  of  the  microscope  a  fully  de- 
veloped colony  is  seen  to  consist  of  a  general  circular 
expansion,  with  thin,  translucent,  smooth,  sharply  de- 
fined margin,  but  becoming  brownish,  granular,  and 
thicker  toward  the  central  portion,  which  is  made  up 
of  coarse,  granular,  brown-colored  clumps  closely  packed 
together. 


298  BACTERIOLOGY. 

The  appearances  coincide  with  the  figure  of  such  a 
colony  given  by  Wertheim.1 

Wassermann  2  calls  attention  to  the  success  he  has 
had  in  cultivating  this  organism  upon  a  mixture  of 
swine-serum  and  nitrose,  the  latter  being  a  com- 
mercial product  chemically  known  as  casein-sodium 
phosphate. 

The  preparation  of  the  medium  and  its  composition 
are  as  follows  : 

In  an  Erlenmeyer  flask  mix  15  c.c.  of  swine-serum,  as 
free  as  possible  from  hemoglobin  ;  30  to  35  c.c.  of  water ; 
2  to  3  c.c.  of  glycerin ;  and  finally  0.8  to  0.9  gramme 
(i.  e.,  about  2  per  cent.)  of  nitrose.  This  is  boiled,  writh 
gentle  agitation,  over  a  free  flame,  until  all  ingredients 
are  dissolved  and  the  cloudy  fluid  has  become  quite 
clear.  After  such  boiling  the  mixture  can  be  sterilized 
by  steam  without  precipitating  the  albumin,  and  may 
then  be  kept  indefinitely  ready  for  use. 

When  needed,  the  flask  and  its  contents  are  heated  to 
50°  C. ;  from  six  to  eight  tubes  of  2  per  cent,  peptone- 
agar-agar  are  dissolved  by  boiling,  brought  to  50°  C., 
and  then  mixed  with  the  solution  in  the  flask  and  the 
mass  poured  into  Petri  dishes.  Upon  the  surface  of  this 
serum-nitrose-agar  the  cultivation  is  to  be  conducted. 
Wassermann  lays  particular  stress  upon  two  points  that 
are  essential  to  success,  viz.,  the  preliminary  boiling  of 
the  serum-nitrose  mixture  before  steam  sterilization,  as 
this  prevents  precipitation  of  the  albumin ;  and  the 
necessity  of  having  both  the  serum-nitrose  mixture  and 
the  agar-agar,  to  be  mixed  with  it,  at  not  over  50°  C., 

1  Deutsche  med.  Wochenschrift,  1891,  No.  50  ;  Centralblatt  fur  Gyna- 
kologie,  1891,  No.  24. 

2  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  Bd.  xvii.  p.  298. 


MICROCOCCUS  GONOREHCEM  299 

for  if  they  are  at  boiling  temperature  when  mixed,  or  if 
they  are  brought  to  the  boiling  temperature  after  mixing, 
the  albumin  will  be  precipitated  notwithstanding  the 
presence  of  the  nitrose,  which  otherwise  prevents  this. 

Wassermann  further  observes  that  some  samples  of 
serum  require  to  be  more  highly  diluted  with  water  than 
in  the  proportions  given  above ;  that  the  agar-agar 
should  be  feebly,  but  distinctly,  alkaline  to  litmus, 
causing  no  reddening  whatever  of  blue  litmus  paper ; 
and,  finally,  that  the  Petri  dishes  containing  the  solidi- 
fied medium  on  which  the  cultures  are  growing  are  best 
kept  bottom  upward,  so  as  to  prevent  water  of  con- 
densation collecting  on  the  surface.  By  the  use  of  the 
above  medium  he  has  cultivated  the  gonococcus  from 
about  one  hundred  different  cases. 

LIPSCHUTZ'S  MEDIUM. — Lipschiitz l  publishes  a  new 
medium  for  the  cultivation  of  micrococcus  gonorrhoese. 
He  sought  to  find  a  medium  that  could  be  prepared 
easily  from  substances  occurring  in  commerce.  After 
testing  a  number  of  albuminous  preparations  of  vege- 
table and  animal  origin,  he  selected  the  pulverized  egg- 
albumen  of  Merck  for  this  purpose.  The  culture- 
medium  is  prepared  as  follows  :  A  2  per  cent,  solution 
of  the  egg-albumin  is  made  in  water,  to  which  is  added 
20  c.c.  of  a  tenth-normal  caustic  soda  solution  per  100 
c.c.  of  fluid,  and  this  is  allowed  to  stand  for  one-half  hour, 
being  agitated  from  time  to  time.  It  is  then  filtered 
and  placed  in  Erlenmeyer  flasks  in  amounts  of  30  to  £0 
c.c.,  and  sterilized  by  the  intermittent  method.  The 
medium,  when  thus  prepared,  is  colorless,  transparent, 
of  a  light-yellow  color,  and  reacts  distinctly  alkaline  to 
iitmus-paper.  To  this  medium  nutrient  agar-agar  or 

1  Lipschiitz :  Centralblatt  fur  Bacteriologie,  Originate,  Bd.  36,  1904. 


300  B  A  CTmiOLOGY. 

the  ordinary  bouillon  may  be  added  in  the  proportion 
of  one  part  of  the  egg-albumin  medium  to  two  or  three 
parts  of  the  agar  medium  or  the  bouillon,  and  this  he 
calls  the  " egg-albumin-agar "  or  the  "egg-albumin- 
bouillon'7  media,  on  which  micrococcus  gonorrhoea  grows 
very  satisfactorily.  The  special  advantages  claimed  for 
this  medium  are  that  it  can  be  prepared  at  any  time  and 
without  difficulty,  is  quite  clear  and  transparent,  and 
permits,  where  agar-agar  is  used,  the  employment  of 
the  medium  for  the  study  of  colony  formations. 

If  transplanted  from  the  original  culture  to  either 
glycerin-agar-agar  or  to  Loffler's  serum-mixture,  a 
growth  is  sometimes  observed,  more  often  in  the  latter 
than  in  the  former,  but  of  so  feeble  a  nature  that  these 
substances  cannot  be  regarded  as  suitable  for  its  culti- 
vation. As  a  rule,  development  does  not  occur  on 
glycerin-agar. 

Microscopic  examination  of  colonies  of  this  organism 
reveals  the  presence  of  a  diplococcus  somewhat  larger 
than  the  ordinary  pyogenic  cocci.  The  opposed  sur- 
faces of  the  individual  cells  that  comprise  the  couplets 
are  flattened  and  separated  by  a  narrow  slit.  At  times 
the  cocci  are  arranged  as  tetrads. 

This  organism  cannot  be  grown  at  a  temperature 
lower  than  that  of  the  human  body,  and  cultures  that 
have  been  obtained  by  either  of  the  favorable  methods 
are  said  to  lose  their  vitality  when  kept  at  ordinary 
room-temperature  for  about  two  days. 

It  is  killed  in  a  few  hours  by  drying. 

Cultures  retain  their  vitality  under  favorable  condi- 
tions of  nutrition,  temperature,  and  moisture  for  from 
three  to  four  weeks. 


MrCROCOCCUS  GONORRHWM  301 

This  organism  is  without  pathogenic  properties  for 
monkeys,  dogs,  and  horses,  as  well  as  for  the  ordinary 
smaller  animals  used  for  this  purpose  in  the  laboratory. 

In  man  typical  gonorrhoea  has  been  produced  on 
several  occasions  by  the  introduction  into  the  urethra 
of  pure  cultures  of  this  organism. 

In  addition  to  its  causal  relation  to  specific  ure- 
thritis,  it  is  the  cause  of  gonorrhoea!  prostatitis  in 
man,  of  gonorrhoea!  proctitis  in  both  sexes,  and  of  gon- 
orrhoea! inflammation  of  the  urethra,  of  Bartholin's 
glands,  of  the  cervix  uteri,  and  of  the  vagina  in 
women  and  young  girls.  It  is  etiologically  related  to  the 
specific  conjunctivitis  (ophthalmia  neonatorum)  of  young 
infants,  and  also  occasionally  to  ophthalmia  in  adults. 

Secondarily,  it  is  concerned  in  specific  inflammations 
of  the  tubes  and  ovaries,  of  the  lymphatics  communi- 
cating with  the  genitalia,  of  the  serous  surfaces  of  joints, 
and  of  those  of  the  heart,  lungs,  and  abdominal  cavity. 

Other  species  of  micrococci  have  from  time  to  time 
been  described  as  occurring  in  the  pus  of  acute  urethritis 
and  of  other  purulent  inflammations.  Many  of  these 
are  of  no  significance.  Some  of  them  possess  peculiarities 
that  might  lead  to  confusion.  The  diplococcus  described 
by  Heiman1  has  certain  points  of  resemblance  to  the 
gonococcus,  such  as  its  location  in  the  bodies  of  pus- 
cells,  its  grouping  as  diplococci,  its  size  and  general  ap- 
pearance ;  but  it  is  still  readily  distinguished  from  the 
gonococcus  by  its  retention  of  color  when  stained  by 
Gram's  method.  The  diplococcus  detected  by  Bumm 
in  puerperal  cystitis  is  likewise  often  found  within  pus- 
cells,  but  it  is  readily  differentiated  from  the  gono- 
coccus by  its  growth  upon  ordinary  nutrient  media. 
1  New  York  Medical  Record,  June  22,  1895. 


30  2  BA  CTP1RIOL  O  G  Y. 

Microcoecus  intraeettularis  of  Weichselbaum,  isolated 
from  the  pus  of  cerebro-spinal  meningitis,  is  micro- 
scopically also  strikingly  like  the  gonococcus  as  it  is 
seen  in  pus ;  but,  unlike  the  latter  organism,  may  be 
cultivated  by  the  ordinary  methods. 

POSITIVE  AND  NEGATIVE  DISTINGUISHING  PECU- 
LIARITIES OF  MICROCOCCUS  GONORRHOEA. — Since  gon- 
orrhoeal  discharges  may  be  contaminated  with  pyogenic 
cocci  other  than  those  causing  the  specific  inflammation, 
it  is  important  in  eiforts  to  isolate  this  organism  that 
the  differential  tests  be  borne  in  mind  and  put  into 
practice.  The  gonococcus  is  differentiated  from  the 
commoner  pyogenic  organisms  by  the  following  pecu- 
liarities : 

First,  it  is  practically  always  seen  in  the  form  of 
diplococci,  the  pair  of  individual  cells  having  the 
appearance  of  two  hemispheres,  with  the  diameters 
opposed,  and  separated  from  one  another  by  a  narrow, 
colorless  slit.  (Is  this  the  case  with  micrococcus  aureus 
or  streptococcus  pyogenes  ?) 

Second,  in  gonorrhoeal  pus  it  is  practically  always  to 
be  found  within  the  protoplasmic  bodies  of  pus-cells. 
(How  does  this  compare  with  the  conditions  found  in 
ordinary  pus  ?) 

Third,  it  stains  readily  with  the  ordinary  staining- 
reagents,  but  loses  its  color  when  treated  by  the  method  of 
Gram.  (Treat  a  cover-slip  from  ordinary  pus  by  this 
method  and  note  the  result.) 

Fourth,  it  does  not  develop  upon  any  of  the  ordinary 
media  used  in  the  laboratory ;  while  the  common  pus- 
organisms,  with  perhaps  the  exception  of  the  strepto- 
cocci, are  vigorous  growers  and  are  not  markedly  fas- 
tidious as  to  their  nutritive  medium. 


MICROCOCCUS  IXTRACELLULARIS.  303 

Fifth,  when  obtained  in  pure  culture  by  either  of  the 
special  procedures  noted  above,  its  cultivation  may  be 
continued  upon  the  same  medium ;  but  growth  will 
usually  not  be  observed  if  it  is  transplanted  to  ordi- 
nary nutrient  gelatin,  agar-agar,  bouillon,  or  potato ; 
should  it  grow  under  these  circumstances  its  develop- 
ment will  be  very  feeble.  (Is  this  the  case  with  com- 
mon pus-producers  ?) 

Sixth,  it  has  no  pathogenic  properties  for  animals, 
while  several  of  the  pyogenic  cocci,  notably  micrococcus 
aureus  and  streptococcus  pyogencs,  are  usually  capable 
of  exciting  pathological  conditions.  (This  is  less  com- 
monly true  of  streptococcus  pyogenes  than  of  micrococcus 
aureus.) 

MICKOCOCCUS    INTRACELLULARIS   (WEICHSELBUAM), 
MIGULA,  1900. 

Synonyms  :  Diplococcus  intracellularis  meningitidis,  Weichselbaum, 
1887;  Streptococcus  intracellularis  (Weichselbaum),  Lehmanu  and 
Neumann,  1896. 

Of  the  several  organisms  mentioned  that  might  be 
mistaken  for  the  gonococcus,  no  one  of  them  is  as  sug- 
gestive and  none,  per  se,  so  important  as  that  concerned 
in  the  causation  of  epidemic  cerebrospinal  meningitis. 

This  organism,  described  by  Weichselbaum  in  1887 
under  the  name  "diplococcus  intracellularis  meningi- 
tidis," was  found  by  him  in  the  exudations  of  the  brain  and 
spinal  cord  in  six  cases  of  acute  cerebrospinal  meningitis. 

As  its  name  implies,  it  is  a  diplococcus,  practically 
always  seen  within  the  bodies  of  pus-cells  (polymorpho- 
nuclear  leucocytes)  in  the  exudations  characteristic  of 
this  disease.  It  is  not  seen  within  the  other  cells  of  the 
morbid  process.  It  stains  readily  with  any  of  the  ordi- 


304  BACTERIOLOGY. 

nary  aniline  dyes,  but  is  decolorized  by  the  method  of 
Gram.  It  is  conspicuous  for  the  irregular  way  in  which 
it  takes  up  the  dye,  some  cells  in  a  preparation  (either 
from  the  exudate  or  from  cultures)  being  brightly  and 
intensely  colored,  others  being  much  less  so,  or,  indeed, 
often  nearly  colorless.  There  is  also  a  marked  variation 
in  the  size  of  individual  cocci,  some  being  normal,  others 
being  apparently  swollen.  These  latter  are  often  pale, 
with  a  deeply  staining  centre,  giving  the  appearance  of 
a  coccus  surrounded  by  a  capsule.  It  is  not  improbable 
that  these  are  degenerated  or  involuted  cells.  The 
irregularities  here  noted  are  more  common  in  cultures 
than  in  fresh  exudates  from  acute  cases,  and  more 
common  in  old  than  in  young  cultures.  As  seen  in 
cultures,  it  is  commonly  arranged  in  pairs  with  the 
individuals  flattened  at  the  surfaces  of  juxtaposition. 
Sometimes  it  is  seen  grouped  as  four  and  occasionally  as 
short  chains  of  three  or  four  cells,  but  never  as  long 
chains.  Its  size  is  that  of  the  common  pyogenic  micro- 
cocci,  and  its  outline  and  arrangement  in  the  pus-cells 
are  so  like  those  of  the  gonococcus  that  the  figure  depict- 
ing gonorrhoaal  pus  answers  equally  well  to  illustrate  the 
appearance  of  the  exudate  from  acute  meningitis. 

While  a  facultative  saprophyte,  still  its  parasitic 
nature  is  so  marked  that  it  can  only  be  cultivated  with 
some  trouble  and  uncertainty.  The  most  satisfactory 
medium  for  its  isolation  in  pure  culture  from  the  dis- 
eased meninges  is  coagulated  blood-serum  (Loffler's 
mixture),  and  even  here  one  is  not  successful  with  each 
attempt.  So  uncertain  is  its  growth  under  artificial 
conditions  that  it  is  always  advisable  to  inoculate  a 
number  of  tubes  with  relatively  large  quantities  of  the 
exudate,  and  even  then  growth  often  occurs  in  only  a 


MICROCOCCUS  INTRA CEL L  ULARIS.  305 

part  of  them,  notwithstanding  the  fact  that  on  micro- 
scopic examination  the  organism  may  have  been  readily 
detected  in  large  numbers  in  the  exudate.  Illustrative 
of  this  difficulty,  the  following  experience  of  Council- 
man, Mallory,  and  Wright  may  properly  be  quoted:1 

"As  showing  the  difficulty  in  growing  the  organisms 
in  cultures  made  from  the  meninges  at  the  post-mortern 
examination,  ten  cultures  were  made  in  one  case  from 
the  exudation  on  the  brain  and  six  from  the  cord,  cover- 
slip  examinations  showing  abundant  organisms  in  the 
cells.  Only  two  of  the  cultures  from  the  brain  and  one 
from  the  cord  showed  a  growth.  As  a  rule,  the  organ- 
isms were  more  easily  obtained  in  cultures  made  from 
the  acute  cases  than  from  the  chronic.'7 

When  successfully  isolated  in  pure  culture  its  growth 
is  never  profuse  on  any  medium.  On  the  serum  mixt- 
ure of  Loffler  the  isolated  colonies  appear  as  round, 
viscid,  smooth,  sharply  defined  points  that  may  attain 
a  diameter  of  1  to  1.5  mm.  There  is  no  liquefaction 
of  the  medium.  Cultures  from  very  acute  cases  occa- 
sionally present  an  abundant  growth  of  fine,  transparent 
colonies  strongly  suggestive  of  those  of  micrococcus 
lanceolatus. 

On  glycerin-agar  the  colonies  are  round,  pearly,  trans- 
lucent, flat,  and  viscid  in  appearance.  They  tend  to 
become  confluent.  Under  low  magnifying  power  thoy 
are  homogeneous,  semitransparent,  faintly  brownish, 
with  well-defined  smooth  margins.  On  plain  agar  the 
growth  is  feeble  and  uncertain. 

Its  growth  in  bouillon  is  slow  and  uncertain.  It  does 
not  cause  clouding  of  the  fluid,  but  collects  at  the  bottom 

1  See  "  Epidemic  Cerebrospinal  Meningitis,"  etc.,  Report  of  the  State 
Board  of  Health,  Mass.,  1898,  by  Councilman,  Mallory,  and  WHght. 

20 


306  B  A  GTE  RIO  L  OGY. 

of  the  tube  as  a  scanty  grayish  sediment,  that  when  dis- 
turbed gives  the  impression  of  having  a  mucoid  con- 
sistency. 

It  does  not  grow  on  potato  and  causes  no  change  in 
litmus-milk. 

It  grows  only  at  the  temperature  of  the  body,  and 
can  be  kept  growing  only  by  being  transplanted  to  fresh 
media  about  every  two  days,  and  even  then  growth 
often  ceases  after  a  comparatively  small  number  of  trans- 
plantations. If  from  a  fresh  growing  culture  a  number 
of  tubes  be  inoculated  and  kept  under  favorable  condi- 
tions, it  is  a  common  experience  to  have  growth  on  only 
a  part  of  them.  It  is  sometimes  impossible  to  obtain 
a  second  growth  on  agar-agar. 

In  addition  to  its  presence  in  the  meningeal  exuda- 
tion of  epidemic  cerebrospinal  meningitis,  this  organism 
may  appear  as  a  secondary  invader  of  the  lung,  causing 
more  or  less  extensive  pneumonic  exudation ;  of  the 
joints ;  the  ear ;  the  eye ;  and  the  nose  and  throat. 
Though  rarely,  its  presence  in  the  circulating  blood  may 
sometimes  be  demonstrated  (Gwynn). 

By  none  of  the  ordinary  methods  of  inoculation  can 
the  disease  be  reproduced  in  animals.  Subcutaneous 
inoculation  with  pure  cultures  has  no  effect.  Injections 
into  the  great  serous  cavities  may  or  may  not  result  in 
serofibrinous  or  fibrinopurulent  inflammation.  Intra- 
venous inoculations  are  equally  unsatisfactory. 

The  only  successful  attempts  to  reproduce  the  morbid 
conditions  from  which  the  organism  is  obtained  are 
those  in  which  the  living  cultures  have  been  injected 
directly  into  the  meninges.  Weichselbaum  produced 
congestion  with  pus  formation  in  the  meninges  of  dogs 
and  rabbits  by  direct  injection  through  openings  made 


MICROCOCCVS  INTRACELLULARIS.  307 

in  the  skulls;  and  Councilman,  Mallory,  and  Wright 
caused  the  death  of  a  goat  by  the  injection  into  the 
spinal  canal  of  1  c.c.  of  a  bouillon  suspension  of  a  pure 
culture  of  the  organism.  The  autopsy  revealed  intense 
congestion  of  the  meninges  of  both  brain  and  cord,  with 
slight  clouding  of  the  meninges  and  slight  increase  of 
meningeal  fluid.  Microscopically  there  was  deep  injec- 
tion of  the  vessels  of  the  cerebral  meninges,  accompanied 
by  an  exudation  composed  principally  of  pus-cells. 
There  was  very  little  fibrin  and  only  small  numbers  of 
diplococci  in  the  pus-cells.  The  purulent  infiltration 
extended  along  the  vessels  into  the  cerebral  cortex. 
The  morbid  condition  was  less  marked  in  the  cord  than 
in  the  brain.  The  micrococci  were  recovered  in  pure 
culture  both  from  cord  and  brain. 

While  the  portal  of  entry  for  this  organism  to  the 
system  is  not  known,  it  is  still  of  importance  to  note 
that  it  often  makes  its  exit  from  the  body  by  way  of  the 
organs  that  are  secondarily  involved,  and  that  open  to 
without,  as  the  ear,  nose,  eye,  and  lungs. 

It  is  of  equal  importance  to  note  that  the  organism  is 
of  very  low  power  of  resistance,  being  destroyed  in 
twenty-four  hours  by  direct  sunlight  and  by  drying  at 
body-temperature,  and  in  seventy-two  hours  by  drying 
in  the  dark  at  ordinary  room-temperature. 

For  the  diagnosis  during  life  of  epidemic  cerebrospinal 
meningitis  by  bacteriological  methods  it  is  desirable  that 
the  meningeal  fluid  be  obtained  during  the  most  acute 
stage  of  the  disease.  This  is  best  done  by  the  operation 
of  lumbar  puncture,  a  description  of  which,  as  given  by 
Mallory  and  Wright,  is  as  follows  : 

"  The  operation  and  the  subsequent  examination  of 
the  fluid  should  be  as  carefully  performed  as  any  other 


308  BA  CTERIOLOG  Y. 

bacteriological  investigation,  in  order  to  obtain  accurate 
results.  The  back  of  the  patient  and  the  operator's 
hands  should  be  made  sterile.  The  needle  should  be 
boiled  for  ten  minutes.  The  patient  should  lie  on  the 
right  side,  with  the  knees  drawn  up,  and  with  the 
uppermost  shoulder  so  depressed  as  to  present  the 
spinal  column  to  the  operator.  This  position  permits 
the  operator  to  thrust  the  needle  directly  forward  rather 
than  from  side  to  side.  An  antitoxin  needle,  4  cm.  in 
length,  with  a  diameter  of  1  mm.,  is  well  adapted  for 
infants  and  young  children.  A  longer  needle  is  neces- 
sary for  adults  and  children  over  ten  years  of  age. 

"Aspiration  of  the  fluid  is  not  necessary,  but  some 
operators  prefer  to  attach  a  hypodermic  syringe  to  the 
needle,  to  aiford  a  better  grasp  for  the  hand.  In  this 
case  the  syringe  would  have  to  be  detached  to  allow 
the  fluid  to  flow.  The  additional  manipulation,  and 
possibly  the  defective  sterilization  of  the  syringe, 
might  impair  the  subsequent  bacteriological  examina- 
tion. 

"  The  puncture  is  generally  made  between  the  third 
and  the  fourth  lumbar  vertebra?,  sometimes  between  the 
second  and  third.  The  thumb  of  the  left  hand  is 
pressed  between  the  spinous  processes,  and  the  point  of 
the  needle  is  entered  about  1  cm.  to  the  right  of  the 
median  line.  Care  must  be  exercised  to  prevent  the 
point  of  the  needle  from  passing  to  the  left  of  the 
median  line  and  striking  the  bone.  At  a  depth  of  3  or 
4  cm.  in  children  and  7  or  8  cm.  in  adults  the  needle 
enters  the  subarachnoid  space,  and  the  fluid  flows  usually 
by  drops.  If  the  point  of  the  needle  meets  with  a  bony 
obstruction,  it  is  advisable  to  withdraw  the  needle  some- 
what, and  to  thrust  again,  directing  the  point  of  the 


PSEUDOMONAS  ^RUGINOSA.  309 

needle  toward  the  median  line,  rather  than  to  make 
lateral  movements,  with  the  danger  of  breaking  the 
needle  or  causing  a  hemorrhage.  The  smallest  quantity 
of  blood  obscures  the  macroscopic  appearance  of  the 
fluid  by  rendering  it  cloudy.  The  fluid  is  allowed  to 
drop  into  an  absolutely  clean  test-tube,  which  previously 
has  been  sterilized  by  dry  heat  to  150°  C.  and  stop- 
pered with  cotton.  The  fluid  should  be  allowed  to 
drop  into  the  tube  without  running  down  the  sides. 
From  5  to  15  c.c.  of  fluid  is  a  sufficient  quantity  for 
examination." 1 

PSEUDOMONAS    ^ERUGINOSA  (SCHROTER,  1872), 
MIGULA,  1900. 

Synonyms  :  Bacterium  eeraginosum,  Schroter,  1872 ;  Bacillus  seru- 
ginosus,  Schroter,  1872;  Bacillus  pyocyaneus,  Gessard,  1882;  Pseudo- 
inouas  pyocyanea,  Migula,  1896. 

Another  common  organism  that  may  properly  be 
mentioned  at  this  place,  though  perhaps  not  strictly 
pyogenic,  is  a  pseudomonas  frequently  found  in  dis- 
charges from  wounds,  viz.,  pseudomonas  ceruginosa,  or 
"  bacillus  of  green  pus,"  or  of  blue  pus,  or  of  blue-green 
pus,  as  it  is  variously  designated.  Pseudomonas  ceru- 
ginosa  is  a  delicate  rod  with  rounded  or  pointed  ends. 
It  is  actively  motile ;  does  not  form  spores.  As  seen 
in  preparations  made  from  cultures,  it  is  commonly  clus- 
tered in  irregular  masses.  It  does  not  form  long  fila- 
ments, there  being  rarely  more  than  four  joined  end  to 
end,  and  most  frequently  occurs  as  single  cells. 

It  grows  readily  on  all  artificial  media,  and  gives  to 

1  For  a  comprehensive  treatment  of  this  subject  from  its  etiological 
and  pathological  standpoints,  see  the  monograph  of  Councilman,  Mai- 
lory,  and  Wright,  to  which  reference  was  made  above,  and  to  which 
we  are  indebted  for  much  contained  in  the  foregoing  sketch. 


310 


BACTERIOLOGY. 


some  of  them  a  bright-green  color  that  is  most  conspic- 
uous where  it  is  in  contact  with  the  air.  This  green 
color,  which  becomes  more  and  more  marked  as  growth 
advances,  is  not  seen  in  the  growth  itself  to  any  extent, 
but  is  diffused  through  the  medium  on  which  the  organ- 
ism is  developing.  Ultimately  this  color  becomes  much 
darker,  and  in  very  old  agar-agar  cultures  may  become 
almost  black  (sometimes  very  dark-blue  green,  at  others 
brownish-black). 


FIG.  58. 


FIG.  59. 


Colony  of  ps.  seruginosa  after  twenty-four  hours 
on  gelatin  at  20°-22°  C. 

FIG.  60. 


J 

Stab-culture  of 
j>*.  smiffinosa  in  gel- 
atin after  twenty- 
eight  hours  at  22°  C. 


Colony  of  p*.  aeruginoma  after  forty-two  hours  on 
gelatin  at  20°-22°  C. 


NOTE. — To  a  fresh  agar  culture  of  this  organism,  in 
which  the  green  coloration  of  the  medium  is  especially 
marked,  add  about  2  c.c.  of  chloroform.  Shake  gently,  and 


rSEUDOMOXAS  ^ERUGINOSA.  311 

note  that  the  chloroform  extracts  a  blue  coloring-matter 
from  the  culture,  leaving  the  latter  more  or  less  yellow. 

Its  growth  on  gelatin  in  stab-cultures  is  accompanied 
by  liquefaction  and  the  diffusion  of  a  bright-green  color 
throughout  the  surrounding  unliquefied  medium.  As 
liquefaction  continues,  and  the  whole  of  the  gelatin 
ultimately  becomes  fluid,  the  green  color  is  confined 
to  the  superficial  layers  in  contact  with  the  air.  The 
form  taken  by  the  liquefying  portion  of  the  gelatin  in 
the  earliest  stages  of  development  is  somewhat  that  of 
an  irregular  slender  funnel.  (See  Fig.  58.) 

On  gelatin  plates  the  colonies  develop  rapidly ;  they 
are  not  sharply  circumscribed,  but  usually  present  at 
first  a  fringe  of  delicate  filaments  about  their  periphery. 
(See  Fig.  59.)  As  growth  progresses  and  liquefaction 
becomes  more  advanced  the  central  mass  of  the  colony 
sinks  into  the  liquid,  while  at  the  same  time  there  is 
an  extension  of  the  colony  laterally.  At  this  stage  the 
colony,  when  slightly  magnified,  may  present  various 
appearances,  the  most  common  being  that  shown  in 
Fig.  60. 

The  gelatin  between  the  growing  colonies  takes  on  a 
bright  yellowish-green  color;  but  as  growth  is  compar- 
atively rapid,  it  is  quickly  entirely  liquefied,  and  one 
often  sees  the  colonies  floating  about  in  the  pale-green 
fluid. 

On  agar-agar  the  growth  is  dry,  sometimes  with  a 
slight  metallic  lustre,  and  is  of  a  whitish  or  greenish- 
white  color,  while  the  surrounding  agar-agar  is  bright 
green.  With  time  this  bright  green  becomes  darker, 
passing  into  blue-green,  and  finally  turns  almost  black. 

On  potato  the  growth  is  brownish,  dry,  and  slightly 


31 2  BA  CTERIOLOG  Y. 

elevated  above  the  surface.  In  some  cultures  tlio 
potato  about  the  line  of  growth  becomes  green ;  in 
others  this  change  is  not  so  noticeable.  With  many 
cultures  a  peculiar  phenomenon,  consisting  of  a  change 
of  color  from  brown  to  green,  may  be  produced  by 
lightly  touching  the  growth  with  a  sterile  platinum 
needle.  The  change  occurs  only  at  the  point  touched. 
It  is  best  seen  in  cultures  that  have  been  kept  in  the 
incubator  for  from  seventy-two  to  ninety -six  hours.  It 
occurs  in  from  one  to  three  minutes  after  touching  with 
the  needle,  and  may  last  from  ten  minutes  to  half  an 
hour.  This  is  the  "  chameleon  phenomenon  "  of  Paul 
Ernst. 

In  bouillon  the  green  color  appears,  and  the  growtli 
is  seen  in  the  form  of  delicate  flocculi.  A  very  delicate 
mycoderma  is  also  produced.  As  growth  progresses, 
the  bouillon  becomes  darker  and  darker  in  color,  until 
it  finally  is  about  comparable  in  this  respect  with  crude 
petroleum ;  at  the  same  time  it  assumes  a  peculiar  ropi- 
ness,  and  very  old  cultures  (four  to  six  weeks  in  the 
incubator)  may  attain  about  the  consistency  of  egg- 
albumin.  This  is  due  to  the  production  of  a  substance 
closely  allied,  chemically  speaking,  to  mucin.  Whether 
it  is  a  metabolic  product  or  one  resulting  from  the 
degeneration  or  the  auto-digestion,  so  to  speak,  of  the 
bacteria,  cannot  now  be  said ;  at  all  events,  in  cultures 
presenting  this  peculiarity  very  few  bacteria  of  normal 
appearance — indeed,  very  few  bacteria  at  all — are  to  be 
seen  on  microscopic  examination. 

In  milk  it  causes  an  acid  reaction,  with  coincident 
coagulation  of  the  casein. 

On  blood-serum  and  egg-albumin  its  growth  is  ac- 
companied by  liquefaction.  The  growth  on  coagulated 


PSP1VDOMONAS  ^ERUGINOSA.  313 


egg-albumin  is  seen  as  a  dirty-gray  deposit  surrounded 
by  a  narrow  brownish  zone  ;  the  remaining  portion  of 
the  medium  is  bright  green  in  color.  As  the  culture 
becomes  older  the  green  may  give  way  to  a  brown  dis- 
coloration. 

In  peptone  solution  (double  strength)  it  causes  a 
bluish-green  color.  In  one  of  four  cultures  from  differ- 
ent sources  we  observed  the  production  of  a  distinct 
blue  color. 

It  produces  indol. 

It  stains  with  the  ordinary  dyes,  and  its  flagella  may 
readily  be  demonstrated  by  Loffler's  method  of  staining. 

It  is  an  active  producer  of  a  proteolytic  enzyme  that 
may  readily  be  separated  and  its  digestive  properties 
observed  by  the  following  simple  method  :  Prepare  a 
bouillon  culture  of  about  70  to  80  c.c.  volume,  and 
allow  it  to  grow  at  37°  to  38°  C.  for  four  or  five  days. 
Filter  through  a  Berkfeld  filter  into  a  sterile  receiver. 
Under  aseptic  precautions  decant  the  filtrate  into  sterile 
test-tubes,  about  7  c.c.  to  each  tube.  Then  under  aseptic 
precautions  make  the  following  tests  :  To  one  tube  add  a 
small  bit  of  hard-boiled  egg  (about  one-half  the  size  of 
a  pea)  and  place  in  an  incubator.  Render  another  tube 
slightly  acid  with  dilute  hydrochloric  acid,  and  add  a 
bit  of  the  white  of  egg  to  it  also.  Do  the  results  differ? 

Heat  another  tube  to  80°  C.  for  fifteen  minutes,  and 
repeat  the  experiment.  Has  the  heating  had  any  effect? 

To  another  tube  add  carbolic  acid  to  the  extent  of  2 
or  3  per  cent.  Is  the  digestive  activity  of  the  solution 
modified  ? 

To  two  ordinary  tubes  of  gelatin  add  carbolic  acid 
until  it  is  present  to  the  extent  of  0.25  per  cent,  in  each 
tube.  Solidify  the  gelatin  in  one  tube  in  the  upright 


•°>  1  4  11. 1 CTKRIOL  O  G  Y. 

position  ;  let  that  in  the  other  remain  fluid.  On  the 
surface  of  the  former  pour  0.5  c.c.  of  the  pyocyaneus 
filtrate,  and  mark  the  point  of  contaet  between  the 
gelatin  and  filtrate.  To  the  other  tube  add  a  similar 
amount  of  the  filtrate,  mix  thoroughly,  and  solidify  in 
a  glass  of  cold  water. 

At  the  end  of  eighteen  to  twenty  hours  note  result. 
Is  it  possible  to  solidify  again  the  gelatin  through  which 
the  filtrate  was  mixed,  by  placing  the  tube  in  cold  water? 

Do  the  activities  of  this  enzyme  suggest  those  of  any 
of  the  enzymes  encountered  in  the  animal  body? 
Which  ?  and  Why  ? 

INOCULATION  INTO  ANIMALS. — As  a  rule,  cultures 
of  this  organism  obtained  directly  from  the  discharges 
of  the  wound  are  capable,  when  introduced  into  ani- 
mals, of  producing  diseased  conditions ;  but  cultures 
kept  on  artificial  media  for  a  long  time  may  in  part,  or 
completely,  lose  this  power. 

When  guinea-pigs  or  rabbits  are  inoculated  subcuta- 
neously  with  1  c.c.  of  virulent  fluid  cultures  of  this 
organism,  death  usually  results  in  from  eighteen  to 
thirty-six  hours.  At  the  seat  of  inoculation  there  are 
found  an  extensive  purulent  infiltration  of  the  tissues 
and  a  marked  zone  of  inflammatory  oedema. 

When  introduced  directly  into  the  peritoneal  cavity 
the  results  are  also  fatal,  and  at  autopsy  a  genuine 
fibrinous  peritonitis  is  found.  There  is  usually  an  ac- 
cumulation of  serum  in  both  the  peritoneal  and  pleural 
cavities.  At  autopsies  after  both  methods  of  inocula- 
tion the  organisms  will  be  found  in  pure  cultures  in 
the  blood  and  internal  viscera. 

When  animals  are  inoculated  with  small  doses  (less 
than  1  c.c.  of  a  bouillon  culture)  of  this  organism  death 


PSEUDOMONAS  ^RLfGINOSA.  315 


may  not  ensue,  and  only  a  local  inflammatory  reaction 
(abscess-formation)  may  be  set  up.  In  these  cases  the 
animals  are  usually  protected  against  subsequent  inocu- 
lation with  doses  that  would  otherwise  prove  fatal. 

Most  interesting  in  connection  with  pseudomonas 
ceruginosa  is  the  fact,  as  brought  out  in  the  experiments 
of  Bouchard,  and  of  Charrin  and  others,  that  its  prod- 
ucts possess  the  power  of  counteracting  the  pathogenic 
activities  of  bacterium  anthracis.  That  is  to  say,  if  an 
animal  be  inoculated  with  a  virulent  anthrax  culture, 
and  soon  after  be  inoculated  with  a  culture  of  pseudo- 
monas ceruginosa,  the  fatal  effects  of  the  former  inocu- 
lation may  be  prevented.  Emmerich  and  Low  '  are 
inclined  to  attribute  this  to  the  direct  bacteriolytic  action 
of  the  enzymes  upon  the  anthrax  bacteria  introduced 
into  the  tissues. 

In  the  literature  upon  the  green-producing  organisms 
that  have  been  found  in  inflammatory  conditions  sev- 
eral varieties  —  believed  to  be  distinct  species  —  have 
been  described  ;  but  when  cultivated  side  by  side  their 
biological  differences  are  seen  to  be  so  slight  as  to  ren- 
der it  probable  that  they  are  but  modifications  of  one 
and  the  same  species. 

BACILLUS    PESTIS,  YERSIN,  1  894. 
THE    BACILLUS    OF    BUBONIC    PLAGUE. 

Before  passing  from  the  subject  of  suppuration  it 
may  not  be  inappropriate  to  call  attention  to  the  light 
that  modern  methods  of  investigation  have  shed  upon 
the  etiology  of  bubonic  plague,  an  epidemic  disease 

1  Miinchener  med.  Wochenschrift,  1898,  No.  40  ;  Centralblatt  fiir 
Bakteriologie  uncl  Parasitenkunde,  1899,  Abt.  i.  No.  1,  p.  33. 


316  BACTERIOLOGY. 

characterized  by  suppuration  of  the  lymphatic  glands, 
and  accompanied  by  a  very  high  rate  of  mortality. 

This  pestilence,  probably  endemic  in  certain  sections 
of  the  Orient,  is  one  of  the  most  conspicuous  epidemic 
diseases  of  history.  Since  early  in  the  Christian  era  epi- 
demics and  pandemics  of  plague  have  made  their  appear- 
ance in  Europe  at  different  times.  During  and  for  a  time 
after  the  Middle  Ages  it  was  more  or  less  frequent  in 
India,  China,  Arabia,  Northern  Africa,  Italy,  France, 
Germany,  and  Great  Britain.  In  history  it  is  variously 
known  as  the  "  Justinian  Plague  "  of  the  sixth  century, 
the  "  Black  Death  "  of  the  fourteenth  century,  and  the 
"  Great  Plague  of  London  "  of  the  seventeenth  century, 
though  it  is  difficult  to  say  to  what  extent  these  pesti- 
lences were  uncomplicated  manifestations  of  genuine 
bubonic  plague.  During  the  existence  of  the  Justinian 
Plague  10,000  people  are  said  to  have  died  in  Con- 
stantinople in  a  single  day,  and  Hecker  estimates  that 
during  the  pandemic  of  the  Black  Death  25,000,000 
people  (a  quarter  of  the  entire  population  of  Europe) 
succumbed  to  the  disease.  During  the  Great  Plague 
of  London  (1664-?65)  the  total  mortality  for  one  year 
was  68,596,  out  of  an  estimated  population  of  460,000 
souls. 

It  is  not  surprising  to  learn  that  it  was  to  guard 
against  the  plague  that  quarantine  regulations  were 
first  established. 

The  first  and  certainly  the  most  exact  information 
up  to  date  concerning  the  cause  and  pathology  of  the 
plague  resulted  from  the  investigations  of  Yersin,  of 
Kitasato,  and  of  Aoyama,  conducted  during  the  epi- 
demic of  1894  in  Hong-Kong,  China;  although  since 
then  numerous  other  investigators  have  made  addi- 


THE  BACILLUS   OF  BUBONIC  PLAGUE.        317 

tional  important  contributions  to  our  knowledge  of 
the  subject.  The  results  of  these  studies  demonstrate 
that  bubonic  plague  is  an  infectious,  not  markedly  con- 
tagious, disease  that  depends  for  its  existence  upon  the 
presence  in  the  tissues  of  a  specific  micro-organism — 
the  so-called  plague  or  pest  bacillus. 

This  organism  is  described  as  a  short,  oval  bacillus, 
usually  seen  single,  sometimes  joined  end  to  end  in  pairs 
or  threes,  less  commonly  as  longer  threads.  It  stains 
more  readily  at  its  ends  than  at  its  centre.  It  is  some- 
times capsulated  ;  is  non-spore-forming ;  is  aerobic,  and 
is  non-motile.  It  is  found  in  large  numbers  in  suppu- 
rating glands,  and  in  much  smaller  numbers  in  the  cir- 
culating blood.  (Fig.  61.) 

It  is  demonstrable  in  cover-slip  preparations  made 
from  the  pus  and  in  sections  of  the  glands  by  the  ordi- 
nary staining-methods. 

Wilm  l  has  found  it  by  culture  methods  in  the  spleen, 
lungs,  liver,  kidneys,  stomach,  walls  of  the  intestine, 
urine,  and  intestinal  contents  of  fresh  cadavers;  and 
during  life  in  the  blood,  expectoration,  fa3ces,  and  urine 
of  patients  sick  of  plague.  He  failed  to  find  it  in  the 
perspiration. 

Yersin  states  that  it  does  not  retain  the  color  when 
treated  by  the  method  of  Gram ;  while  Kitasato  says 
that  it  at  one  time  stains  by  this  method  and  at  another 
it  becomes  decolorized.  Aoyama  observed  that  those 
bacilli  within  the  suppurating  glands  were  decolorized, 
while  those  in  the  blood  retained  the  stain  when  treated 
by  Gram's  method. 

Since  there  is  often  a  mixed  infection  in  these  cases, 
it  appears  likely  that  the  above  discrepancy  may  be 

1  Wilm:  Hygiciiisuhc  Kundsclmu,  181)7,  p.  217. 


318 


BACTERIOLOGY. 


attributed  to  individual  peculiarities  of  different  species 
of  bacteria  that  were  under  examination,  an  opinion 
that  is  borne  out  by  more  recent  studies,  from  which  it 
has  been  decided  that  the  genuine  plague  or  pest  bacillus 
does  not  stain  by  Gram's  method. 

It  may  be  cultivated  upon  ordinary  nutrient  media, 


Bacillus  of  bubonic  plague :  A,  in  pus  from  suppurating  bubo ;  B,  the 
bacillus  very  much  enlarged  to  show  peculiar  polar  staining. 

although  preference  is  given  by  some  to  a  neutral  or 
slightly  alkaline  2  per  cent,  peptone  solution  containing 
from  1  to  2  per  cent,  of  gelatin. 

The  most  favorable  temperature  for  its  growth  is 
between  36°  and  39°  C.  Its  colonies  on  glycerin-agar- 
agar  and  on  coagulated  blood-serum  are  described  as 


THE  BACILLUS  OF  BUBONIC  PLAGUE.        319 

iridescent,,  transparent,  and  whitish.  On  gelatin  at 
18°— 20°  C.  it  develops  as  small,  sharply  defined,  white 
colonies  without  liquefaction  of  the  medium.  In  stab- 
cultures  it  develops  both  on  the  surface  and  along  the 
track  of  the  needle.  Its  growth  is  slow.  It  does  not 
cause  a  diffuse  clouding  of  bouillon,  but  grows  rather 
as  irregular,  flocculent  clumps  that  adhere  to  the  sides 
or  sink  to  the  bottom  of  the  vessel,  leaving  the  fluid 
clear.  It  shows  but  limited  growth  on  potato.  It  does 
not  ferment  glucose  with  production  of  gas,  nor  does  it 
form  indol.  It  coagulates  milk. 

This  organism  is  killed  by  drying  at  ordinary  room- 
temperature  in  four  days.  It  is  killed  in  three  or  four 
hours  by  direct  sunlight.  It  is  destroyed  in  a  half  hour 
by  80°  C.,  and  in  a  few  minutes  by  100°  C.  (steam). 
It  is  killed  in  one  hour  by  1  per  cent,  carbolic  acid 
and  in  two  hours  by  1  per  cent,  milk  of  lime.1 

It  is  pathogenic  for  rats,  mice,  guinea-pigs,  rabbits, 
hogs,  horses,  monkeys,  cats,  chickens,  and  sparrows. 
Pigeons,  hedgehogs,  and  frogs  are  immune,  and  dogs 
and  bovines  are  apparently  so.2  Animals  succumb  to 
subcutaneous  inoculation  in  from  two  to  three  days. 
According  to  Yersin,  the  site  of  subcutaneous  inocu- 
lation becomes  oedematous  and  the  neighboring  lym- 
phatics are  enlarged  in  a  few  hours.  After  twenty-four 
hours  the  animal  is  quiet,  the  hair  is  rumpled,  tears 
stream  from  the  eyes,  and  later  convulsions  set  in,  which 
last  till  death.  The  results  found  at  autopsy  are  :  blood- 
stained oedema  at  the  site  of  inoculation,  reddening  and 

1  See  "  Viability  of  the  Bacillus  Pestis,"  by  M.  J.  Eosenau,  U.  S. 
Marine-Hospital  Service,  Bulletin  No.  4,  of  the  Hygienic  Laboratory, 
U.  S.  M.-H.,  Washington,  D.  C.,  1901. 

2Nuttall:  Centralblat  fiir  Bakteriologie  und  Parasitenkumle,  18M7, 
Abt.  i.  Bel.  xxii.  S.  1)7. 


320  BACTERIOLOGY. 

swelling  of  the  lymphatic  glands,  bloody  extravasation 
into  the  abdominal  walls,  serous  effusion  into  the  pleu- 
ra! and  peritoneal  cavities ;  the  intestine  is  occasionally 
hypersemic,  the  adrenal  bodies  congested,  and  the  spleen 
enlarged,  often  being  studded  with  grayish  points,  sug- 
gestive of  miliary  tubercles.  The  plague,  or  pest,  ba- 
cillus is  detected  in  large  numbers  in  the  local  oedema, 
the  lymph-glands,  the  blood,  and  the  internal  organs. 

As  is  the  case  in  general  with  the  group  of  hemor- 
rhagic  septicaemia  bacteria,  the  members  of  which  it 
resembles  in  certain  other  respects,  when  death  does  not 
result  promptly  after  infection  there  is  usually  only 
local  evidence  of  the  inoculation,  the  distribution  of  the 
micro-organisms  throughout  the  body  being  considera- 
bly diminished. 

Animals  that  survive  inoculation  with  this  organism 
usually  exhibit  a  certain  degree  of  immunity  from  sub- 
sequent infection. 

Nuttall l  notes  that  feeding-experiments  have  resulted 
in  fatal  infection  in  gray  and  white  rats,  house-  and 
field-mice,  guinea-pigs,  rabbits,  hogs,  apes,  cats,  chick- 
ens, sparrows,  and  flies.  He  also  calls  attention  to  the 
fact  that  flies  may  live  for  several  days  after  being  in- 
fected with  this  organism,  and  if  at  liberty  to  fly  about 
may  manifestly  infect  persons  or  food-stuffs  on  which 
they  alight  or  fall. 

The  bacilli  apparently  lose  their  virulence  after 
long-continued  cultivation  under  artificial  conditions, 
and  it  is  said  that  from  slowly  developing,  chronic 
buboes  non-virulent  or  feebly  virulent  cultures  are 
often  obtained.  Variations  in  the  degree  of  virulence 
have  been  observed  in  different  colonies  from  the  same 

1  Nuttall :  loc.  cit. 


THE  BACILLUS  OF  BUBONIC  PLAGUE.        321 

source.  Virulence  is  said  by  Yersin,  Calmette,  and 
Borrel1  to  be  accentuated  by  passing  the  organism 
through  a  series  of  susceptible  animals. 

In  man  the  bacilli  are  most  numerous  in  the  en- 
larged, suppurating  lymphatics.  They  are  present,  but 
in  smaller  numbers,  in  the  blood  and  the  internal 
organs. 

It  has  been  observed  that  in  the  suppurating  lym- 
phatic glands  of  man  a  variety  of  organisms  may  be 
present,  but  among  them  are  always  the  plague  bacilli. 
Occasionally,  micrococci  predominate.  In  these  cases 
of  mixed  infection  the  pest  bacilli  are  said  to  stain  less 
intensely  with  alkaline  methylene-blue  than  do  the 
streptococci,  and  more  intensely  than  do  the  staphylo- 
cocci  that  are  present.  Also,  in  this  event,  the  strepto- 
cocci retain  the  Gram  stain,  while  the  pest  bacilli  do  not 
and  the  staphylococci  may  or  may  not.  It  has  been 
suggested  that  possibly  the  organisms  found  by  Kitasato 
in  the  blood,  and  which  he  describes  as  pest  bacilli, 
that  retained  the  color  when  treated  by  the  method  of 
Gram,  were  pairs  of  micrococci,  and  not  bacilli  at  all. 

It  is  the  opinion  of  Aoyama  that  the  suppuration  of 
the  glands  is  not  caused  by  the  plague  bacillus,  but  is 
rather  the  result  of  the  action  of  the  pyogenic  cocci 
with  which  it  is  so  often  associated.  It  is  also  his 
belief  that  the  most  important  and  frequent  mode  of 
infection  in  man  is  through  wounds  of  the  skin.  He 
does  not  regard  either  the  air-passages  or  the  alimentary 
tract  as  frequent  portals  of  infection.  Wilm,  on  the 
contrary,  is  inclined  to  regard  the  alimentary  tract  as  a 
frequent  portal  of  infection  ;2  and  subsequent  investiga- 

1  Annales  de  1'Institut  Pasteur,  1895,  p.  589. 

2  Wilm  :  loc.  cit. 
21 


322  BA  CTERIOLOG  Y. 

lions  leave  little  doubt  that  infection  occasionally  occurs 
through  the  respiratory  tract. 

The  order  in  which  the  lymphatics  manifest  disease 
appears  to  depend  upon  the  location  of  the  primary 
infection.  That  is  to  say,  if  it  is  upon  the  feet,  as  of 
persons  who  go  barefooted,  the  superficial  and  deep 
inguinal  glands  are  the  first  to  show  signs  of  the  dis- 
ease ;  while  if  infection  occurs  through  wounds  of  the 
hand,  the  buboes  appear  first  in  the  axillary  region. 
As  a  rule,  the  wound  through  which  infection  is  re- 
ceived shows  little  or  no  inflammatory  reaction.1 

Wyssokowitz  and  Zabolotny2  call  attention  to  the 
fact  that  the  blood  of  patients  convalescing  from  plague 
has  an  agglutinating  action  upon  fluid  cultures  of  the 
plague  bacillus  analogous  to  that  observed  when  the 
blood-serum  of  typhoid  or  of  cholera  patients  is  mixed 
with  similar  cultures  of  the  typhoid  or  the  cholera 
bacillus. 

Yersin,  Calmette,  and  Borrel 3  have  demonstrated  that 
the  general  principles  underlying  the  establishment  of 
artificial  immunity  apply  as  well  to  this  disease  as  to 
a  number  of  others.  They  have  shown  that  by  the  use 
of  dead  cultures  (destroyed  by  heat)  of  the  plague 
bacillus  animals  may  be  rendered  immune  from  infec- 
tion by  the  virulent  living  organism.  They  have  also 
shown  that  the  serum  of  the  blood  of  these  animals  is 
not  only  capable  of  conferring  immunity  upon  other  ani- 
mals into  which  it  is  injected,  but  it  has  curative  proper- 

1  The  works  of  Yersin,  of  Kitasato,  and  of  Aoyama  have  been  ex- 
haustively reviewed  by  Flexner  in  the  Bulletin  of  the  Johns  Hopkins 
Hospital,  1894,  vol.  v.  p.  96,  and  1896,  vol.  vii.  p.  180.     I  am  indebted 
to  these  reviews  for  much  that  is  here  presented  on  this  subject. 

2  Annales  de  1'Institut  Pasteur,  1897,  p.  663. 

3  Ibid.,  1895,  p.  589. 


ANTI-PLAGUE  SERUM.  323 

ties  as  well,  providing  it  be  employed  at  an  early  stage 
of  the  disease.  In  1896  Yersin  l  used  the  serum  of  arti- 
ficially immunized  horses  in  the  treatment  of  plague 
in  human  beings.  Of  26  persons  (3  in  Canton  and  23 
in  Amoy,  China)  who  received  injections  of  the  serum 
during  the  early  stages  of  the  disease,  in  no  case  later 
than  the  fifth  day,  only  2  died.  Comparing  this  mor- 
tality of  7.6  per  cent,  with  the  mortality  of  80  per  cent, 
among  persons  in  this  epidemic  who  were  treated  in 
other  ways,  he  feels  justified  in  regarding  the  method  as 
worthy  of  consideration. 

ANTI-PLAGUE  SERUM. — During  recent  years  a  great 
deal  of  further  investigation  has  been  made  in  order  to 
ascertain  the  pathogenic  properties  of  bacillus  pestis, 
and  to  obtain  a  more  detailed  knowledge  of  immunity 
against  infection  by  this  organism.  The  studies  of  the 
latter  class  include  the  estimation  of  the  quantitative 
value  of  the  pest-serum  when  tested  on  different  species 
of  animals,  its  protective  and  curative  properties  when 
employed  on  such  animals,  especially  its  protective 
properties  against  experimental  inhalation  pneumonia 
and  infection  by  feeding  pi  ague- infected  materials. 
These  observations  have  been  carried  out  by  several 
pest  commissions — namely,  those  of  Germany,  Austria, 
and  Egypt — as  well  as-  the  detailed  investigations  of  the 
Institutes  for  Infectious  Diseases  at  Berlin,  at  Berne, 
and  the  Pasteur  Institute  at  Paris.2  The  preparation 
of  the  pest-serum  is  conducted  as  follows :  Horses  are 
injected  at  first  with  dead  cultures  of  bacillus  pestis, 
then  with  increasing  doses  of  living  agar  cultures  of  the 

1  Annales  de  1'Institut  Pasteur,  1897,  p.  81. 

2  The  important  literature  bearing  on  this  subject  is  appended  to 
the  report  of  Kolle,  Hetsch,  and  Otto  (Zeitschr.  f.  Hygiene,  Bd.  38, 
p.  368). 


324  BACTERIOLOGY. 

highly  virulent  bacillus.  These  organisms  are  injected  at 
intervals  of  from  eight  to  twelve  days.  By  means  of  this 
treatment  a  serum  is  obtained  which  is  of  limited  cura- 
tive value,  but  of  very  marked  protective  value.  Poly- 
valent serums  do  not  seem  to  give  any  better  results. 
Kolle  questions  whether  it  will  ever  be  possible  to  pre- 
pare antitoxic  pest-serum,  because  so  far  we  are  unable 
to  demonstrate  a  true  pest-toxin. 

The  unsatisfactory  results  with  serum  therapy  in 
plague  in  a  number  of  epidemics  cannot  be  attributed, 
according  to  the  investigations  of  Hetsch  and  Ilimpau, 
to  the  absence  of  specific  amboceptors  in  the  pest-serum 
for  bacillus  pestis.  Even  in  the  earlier  investigations 
on  the  estimation  of  the  value  of  the  pest-serum,  Kolle 
was  lead  to  doubt  whether  the  serum  conformed  exactly 
with  the  laws  of  bactericidal  serums ;  in  other  words, 
whether  the  pest-serum  acted  as  a  purely  bactericidal 
serum  or  not.  It  is  true  that  some  bacteriolytic  action 
can  be  demonstrated  in  the  pest-serum  ;  but  Markl  was 
able  to  show  that  with  virulent  cultures  the  leucocytes 
play  a  most  important  part  in  the  destruction  of  the 
bacteria  when  injected  into  the  peritoneal  cavity  of  an 
animal.  Kolle  concludes,  therefore,  that  the  pest-serum 
is  neither  a  purely  antitoxic  serum,  like  that  of  diph- 
theria and  tetanus,  nor  a  purely  bactericidal  serum,  like 
that  of  cholera  and  typhoid,  but  that  its  action  rests 
probably  much  more  upon  substances  the  biological 
character  of  which  is  as  yet  undetermined,  in  addition 
to  some  bactericidal  action.  In  this  respect  it  corre- 
sponds somewhat  with  the  anthrax  serum,  as  shown  by 
the  experiments  of  Sobernheim.  For  this  reason  Kolle 
calls  the  pest-serum  an  "  anti-infectious  "  serum,  as  he 
believes  that  by  this  designation  its  biological  action  is 


THE  HAFFKINE   VACCINE  AGAINST  PLAGUE.    325 

expressed  more  definitely  than  by  the  term  bactericidal 
serum. 

THE  HAFFKINE  VACCINE  AGAINST  PLAGUE. — A 
great  deal  of  work  has  also  been  done  in  recent  years 
in  conferring  active  immunity  against  bacillus  pestis  by 
the  Haff kine  method — that  is,  by  the  injection  of  dead 
cultures  of  the  organism,  and  also  by  the  injection  of 
organisms  of  low  degree  of  virulence,  whereby  an  active 
immunity  is  conferred.  Especially  satisfactory  immuniz- 
ing results  have  'been  obtained  by  combining  the  vacci- 
nation with  dead  cultures  or  cultures  of  low  virulence 
with  the  administration  of  the  immune  serum.  While 
the  vaccination  is  entirely  harmless  for  animals  suscep- 
tible to  bacillus  pestis,  even  in  large  doses,  the  experi- 
ments on  human  beings  could  only  be  carried  out  in 
combination  with  the  administration  of  the  immune 
serum  or  after  previous  injection  of  dead  cultures. 

In  1897  Haifkine  reported  to  the  Indian  government 
that  he  had  prepared  an  inoculation  fluid  for  the  protec- 
tion of  human  beings  against  plague  infection.  This 
fluid  is  now  prepared  by  the  Plague  Research  Labora- 
tory in  Bombay,  and  up  to  December,  1900,  they  had 
distributed  1,628,696  doses.  It  has  been  found  that 
this  fluid  does  not  give  the  same  degree  of  protection 
against  plague  as  does  vaccinia  against  smallpox,  but 
the  general  results  obtained  in  India  indicate  that  the 
individuals  vaccinated  with  this  material  are  not  only 
far  less  liable  to  infection  by  the  plague  organism,  but 
if  they  become  infected  they  are  more  likely  to  recover. 

Bannerman,  of  the  Plague  Research  Laboratory  in 
Bombay,  gives  several  instances  of  the  value  of  the 
inoculation  as  a  protection  against  plague  infection,  and 
cites  the  personnel  of  the  Southern  Mahratta  Railway, 
which  were  as  follows  : 


326  BA  CTERIOLOG  Y. 

Of  990  persons  inoculated  twice,  6  contracted  plague, 
of  which  1,  or  0.1  -j-  per  cent,  of  those  inoculated,  died. 

Of  270  persons  inoculated  once,  5  contracted  plague, 
and  1,  or  0.3+  per  cent,  of  those  inoculated,  died. 

Of  760  persons  not  inoculated,  35  contracted  plague, 
and  21,  or  2.7+  per  cent,  of  those  not  inoculated,  died. 

These  results  indicate  a  reduction  in  the  mortality  of 
94.1  per  cent,  in  those  who  had  been  inoculated. 

In  Hubli  there  were,  in  the  summer  of  1898,  24,631 
inoculated,  compared  with  17,786  uninoculated  persons. 
In  that  city  the  mortality  from  plague  for  the  inoculated 
was  89.6  per  cent,  lower  than  for  the  uninoculated,  and 
Bannerman  states  that  in  practically  all  instances  a 
reduction  in  mortality  of  about  90  per  cent,  is  brought 
about  by  the  anti-plague  vaccination. 

Porsyth 1  reports  on  30,609  cases  in  India  that  had 
been  vaccinated  by  the  HafFkine  method,  of  which 
number  329  were  subsequently  attacked  by  plague,  50 
of  whom  died,  a  case  mortality  of  15.1  per  cent.  He 
gives  a  table  showing  the  relative  behavior  of  uninocu- 
lated and  inoculated  persons  toward  plague. 

M  r-oooo  r>f   Attack-  Death-    Case  mor- 

Classes.  pooh         iSne     rate  per  Deaths-  rate  per   tality  Per 

each.        plague.      cent  cent  cent 

(a)  Uninoculated,  31,874       1457        4.5        659         2.06        45.2 

(b)  Inoculated,       12,886         171         1.3          29         0.22        16.9 

The  anti-plague  vaccine  is  administered  in  doses  of 
5  c.c.  The  reaction  following  the  inoculation  differs  in 
different  individuals,  especially  with  regard  to  the  tem- 
perature. Haffkine  recommends  for  immunizing  pur- 
poses the  employment  of  smaller  doses  for  the  first 
injection,  and  after  the  subsidence  of  the  reaction  the 
use  of  a  larger  second  injection.  Even  under  these 
Conditions  the  reactions  are  sometimes  quite  marked. 
1  Forsyth  :  The  Lancet,  vol.  ii.,  1903,  p.  1646. 


CHAPTER    XVI. 

Sputum  septicaemia— Septicaemia  resulting  from  the  presence  of  sarcina 
tetragena,  or  from  bacterium  pueumonise  in  the  sputum  of  appar- 
ently healthy  persons — The  occurrence  of  bacterium  influenza? 
in  the  sputum. 

IT  is  not  infrequent  that  apparently  healthy  persons 
harbor  in  the  mouth  cavity,  nose,  or  throat  a  variety  of 
pathogenic  organisms  without  manifesting  any  symptoms 
of  their  presence.  Some  of  these  pathogenic  organisms 
may  be  readily  detected  by  appropriate  methods  of  cul- 
tivation. This  is  especially  true  of  the  bacterium  diph- 
theria, as  will  be  shown  in  a  subsequent  chapter.  Some 
of  the  other  pathogenic  bacteria  are  not  so  readily  isolated 
by  the  cultural  method,  but  may  be  demonstrated  by  ap- 
propriate methods  of  staining.  On  staining  cover-glass 
preparations  of  sputum  by  the  Gram  method  and  counter- 
staining  with  eosin  it  is  often  possible  to  detect  bacterium 
pneumonia  and  bacterium  influenza  by  reason  of  their 
peculiar  morphology  and  staining  reactions. 

The  most  satisfactory  results,  however,  are  obtained 
by  the  subcutaneous  or  intravenous  injection  of  the 
sputum  into  guinea-pigs  or  rabbits.  By  this  means  the 
non-pathogenic  organisms  are  quickly  eliminated  and 
the  pathogenic  organisms,  if  present,  produce  their 
characteristic  lesions.  Probably  the  most  frequent  re- 
sult of  such  inoculation  of  sputum  is  the  production  of 
a  general  septicaemia.1 

Obtain  from  a  tuberculous  patient  a  sample  of  fresh 

1  Septicaemia  is  that  form  of  infection  in  which  the  blood  is  the  chief 
field  of  activity  of  the  organisms, 

327 


328  BA  CTERIOL  O  G  Y. 

sputum — that  of  the  morning  is  preferable.  Spread 
it  in  a  thin  layer  upon  a  black  glass  plate  and  select 
one  of  the  small,  white,  cheesy  masses  or  dense  mu- 
cous clumps  scattered  through  it.  With  a  pointed 
forceps  smear  this  carefully  upon  two  or  three  thin 
cover-slips,  dry  and  fix  them  in  the  way  given  for 
ordinary  cover-slip  preparations.  Stain  one  in  the 
ordinary  way  with  Loffler's  alkaline  methylene-blue 
solution,  one  other  by  the  Gram  method,  and  a  third 
after  the  method  given  for  bacterium  tuberculosis  in 
fluids  or  sputum. 

In  that  stained  by  Loffler's  method — slip  No.  1 — 
will  be  seen  a  great  variety  of  organisms — round  cells, 
ovals,  short  and  long  rods,  perhaps  spiral  forms.  But 
not  infrequently  will  be  seen  diplococci  having  more 
or  less  of  a  lancet  shape,  joined  together,  by  their 
broad  ends,  the  points  of  the  lancet  being  away  from 
the  point  of  juncture  of  the  two  cells.  There  may 
also  be  seen  masses  of  cocci  which  are  conspicuous  by 
their  arrangement  into  groups  of  fours,  the  adjacent 
surfaces  being  somewhat  flattened.  They  are  not  sar- 
cinse,  as  one  can  see  by  the  absence  of  the  division  in 
the  third  direction  of  space — they  divide  in  only  two 
directions. 

In  the  slip  stained  by  the  Gram  method  the  same 
groups  of  cocci  which  grow  as  threes  and  fours 
will  be  seen;  but  the  lancet-shaped  diplococci  now 
present  an  altered  appearance — they  are  usually  sur- 
rounded by  a  capsule.  This  capsule  is  very  deli- 
cate in  structure,  and,  though  a  frequent  accompani- 
ment, is  not  constant.  It  can  sometimes  be  demon- 
strated by  the  ordinary  methods  of  staining,  though 
the  method  of  Gram  is  most  satisfactory.  (Fig.  63.) 


SPUTUM  SEPTICAEMIA.  329 

In  the  third  slip,  which  has  been  stained  by  the 
method  given  for  tubercle  bacteria  in  sputum,  if  decolor- 
ization  has  been  properly  conducted  and  no  contrast- 
stain  has  been  employed,  the  field  will  be  colorless  or 
of  only  a  very  pale  rose  color.  None  of  the  numerous 
organisms  seen  in  the  first  slip  can  now  be  detected ; 
but  instead  there  will  be  seen  scattered  through  the 
field  very  delicate  stained  rods,  which  present,  in 
most  instances,  a  conspicuous  beading  of  their  pro- 
toplasm— that  is,  the  staining  is  not  homogeneous, 
but  at  tolerably  regular  intervals  along  each  rod  are 
seen  alternating  stained  and  unstained  points.  These 
rods  may  be  found  singly,  in  groups  of  twos  and  threes, 
and  sometimes  in  clumps  consisting  of  large  numbers. 
When  in  twos  or  threes  it  is  not  uncommon  to  find 
them  describing  an  X  or  a  V  in  their  mode  of  arrange- 
ment, or  again  they  may  be  seen  lying  parallel  the  one 
to  the  other. 

If  contrast-stains  are  used,  these  rods  will  be  detected 
and  recognized  by  their  retaining  the  original  color 
with  which  they  have  been  stained ;  whereas  all  other 
bacteria  in  the  preparation,  as  well  as  the  tissue-cells 
which  are  in  the  sputum,  will  take  up  the  contrast- 
color.  (Fig.  62.) 

This  delicate,  beaded  rod  is  bacterium  tuberculosis. 
The  lancet-shaped  diplococcus  with  the  capsule  is  bac- 
terium pneumonia.  The  cocci  grouped  in  fours  are 
sarcina  tetragena. 

INOCULATION  EXPERIMENT. — Inoculate  into  the 
subcutaneous  tissues  of  a  guinea-pig  one  of  the  small, 
white,  caseous  masses,  similar  to  that  which  has  been 
examined  microscopically.  If  death  ensue,  it  will,  in 


330  BACTERIOLOGY. 

all  probability,  be  the  result  of  one  of  the  three  follow- 
ing forms  of  infection : 

a.  Septicaemia   resulting   from  the  introduction  into 
the  tissues  of  bacterium  pneumonice. 

b.  A  form  of  septicaemia  resulting  from  the  introduc- 
tion of  sarcina  tetragena,  an  organism  frequently  seen  in 
the  sputum  of  tuberculous  subjects. 

c.  Local  or  general  tuberculosis. 

FIG.  62. 


Tuberculous  sputum  stained  by  Gabbett's  method.    Tubercle  bacteria  seen 
as  red  rods ;  all  else  is  stained  blue. 

SPUTUM    SEPTICAEMIA. 

BACTERIUM    PNEUMONIA   (WEICHSELBAUM),  MIGULA, 
1900. 

Synonyms:  Diplococcus  pneumonise,  Weichselbaum,  1886;  Pneumo- 
coccus,  Frankel,  1886 ;  Micrococcus  of  sputum  septicaemia  ;  Diplococcus 
lanceolatus ;  Streptococcus  lanceolatus ;  Streptococcus  pasteuri ;  Micro- 
coccus  lanceolatus. 

If  at  the  end  of  twenty-four  to  thirty-six  hours  the 
animal  be  found  dead,  we  may  reasonably  predict  that 
the  result  was  produced  by  the  introduction  into  the  tis- 
sues of  the  organism  of  sputum  septicaBmia  above  men- 
tioned, viz.,  bacterium  pneumonice,  which  is  not  uncom- 


BACTERIUM  PXEUMONLV.  331 

monly  found  in  the  mouths  of  healthy  individuals  as 
well  as  in  other  conditions. 

Inspection  of  the  seat  of  inoculation  usually  reveals 
a  local  reaction.  "  This  may  be  of  a  serous,  fibrinous, 
hemorrhagic,  necrotic,  or  purulent  character.  Fre- 
quently we  may  find  combinations  of  these  conditions, 
such  as  fibre-purulent,  fibrino-serous,  or  sero-hemo^ 
rhagic." 1  The  most  conspicuous  naked-eye  change 
undergone  by  the  internal  organs  will  be  enlargement 
of  the  spleen.  It  is  usually  swollen,  but  may  at  times 
be  normal  in  appearance.  It  is  sometimes  hard,  dark 
red,  and  dry;  or  it  maybe  soft  and  rich  in  blood.  Fre- 
quently there  is  a  limited  fibrinous  exudation  over  por- 
tions of 'the  peritoneum. 

Except  in  the  exudations,  the  organisms  are  found 
only  in  the  lumen  of  the  bloodvessels,  where  they  are 
usually  present  in  enormous  numbers.  In  the  blood 
they  are  practically  always  free,  and  are  but  rarely  found 
within  the  bodies  of  leucocytes. 

In  stained  preparations  from  the  blood  and  exudates 
a  capsule  is  not  infrequently  seen  surrounding  the  organ- 
isms. (Fig.  63.)  This,  however,  is  not  constant. 

If  a  drop  of  blood  from  the  dead  animal  be  intro- 
duced into  the  tissues  of  a  second  animal  (mouse  or 
rabbit),  identically  the  same  conditions  will  be  repro- 
duced. 

If  the  organism  be  isolated  in  pure  culture  from  the 
blood  of  the  animal,  and  a  portion  of  this  culture  be 
introduced  into  the  tissues  of  a  susceptible  animal, 
we  shall  see  again  the  same  pathological  picture. 

It   must    be    remembered,    however,    that    this   or- 

1  Welch  :  Johns  Hopkins  Hospital  Bulletin,  December,  1892,  vol.  iii. 
No.  27. 


332  BACTERIOLOGY. 

ganism  when  cultivated  for  a  time  on  artificial  media 
rapidly  loses  its  pathogenic  properties.  If,  therefore, 
failure  to  reproduce  the  disease  after  inoculation 
with  old  cultures  should  occur,  it  is  in  all  probability 
due  to  a  loss  of  virulence  of  the  organism. 

FIG.  63. 


f    , 


Bacterium  pneumonice  in  blood  of  rabbit.    Stained  by  method  of  Gram. 
Decolorization  not  complete. 


This  organism  was  discovered  by  Sternberg  in  1880. 
It  was  subsequently  described  by  A.  Friinkel  as  the 
etiological  factor  in  the  production  of  acute  fibrinous 
pneumonia. 

It  is  not  uncommonly  present  in  the  saliva  of  healthy 
individuals,  having  been  found  by  Sternberg  in  the  oral 
cavities  of  about  20  per  cent,  of  healthy  persons  examined 
by  him,  and  certain  authors  are  of  the  opinion  that  it 
occurs  in  the  oral  or  nasal  cavities  of  all  individuals 
at  various  times  during  life.  It  is  constantly  to  be 
detected  in  the  rusty  sputum  of  patients  suffering  from 
acute  fibrinous  pneumonia.  Its  presence  has  been  de- 
tected in  the  middle  ear,  in  the  pericardial  sac,  in  the 
pleura,  and  in  the  serous  cavities  of  the  brain;  and 


BACTERIUM  PNEUMONIA.  333 

indeed  it  may  penetrate  from  its  usual  site  of  develop- 
ment in  the  mouth  to  any  of  the  more  distant  organs. 

The  organism  is  commonly  found  as  a  diplococcus, 
though  here  and  there  short  chains  of  four  to  six  indi- 
viduals may  be  detected.  (Fig.  63.)  The  individual 
cells  are  more  or  less  oval,  or,  more  strictly  speaking, 
lancet-shaped,  for  at  one  end  they  are  commonly  pointed 
When  joined  in  pairs  the  junction  is  always  at  the 
broad  ends  of  the  ovals,  never  at  the  pointed  extremities. 
When  in  chains  only  the  terminal  cells  are  pointed,  and 
then  at  their  distal  extremities. 

As  already  stated,  in  preparations  directly  from  the 
sputum  or  from  the  blood  of  animals  a  delicate  capsule 
may  frequently  be  seen  surrounding  them.  Though 
fairly  constant  in  preparations  directly  from  the  blood 
of  animals  and  from  the  sputum  or  lungs  of  pneumonic 
patients,  the  capsule  is  but  rarely  observed  in  artificial 
cultures.  Occasionally  in  cultures  on  blood-serum,  in 
milk,  and  on  agar-agar  it  can,  according  to  some 
authors,  be  detected ;  but  this  is  by  no  means  constant, 
or  even  frequent. 

Even  under  the  most  favorable  artificial  conditions 
this  organism  grows  but  slowly  and  frequently  not  at 
all. 

When  successfully  grown  upon  the  different  media  it 
presents  somewhat  the  following  appearances  : 

On  gelatin  its  development  is  very  limited  and  often 
no  growth  at  all  occurs.  This  is  probably  due  in  part 
to  the  low  temperature  at  which  gelatin  cultures  must 
be  kept.  If  development  occurs,  the  growth  appears 
as  minute  whitish  or  blue-white  points  on  the  plates. 
These  very  small  colonies  are  round,  finely  granular, 
sharply  circumscribed,  and  slightly  elevated  above  the 


334  SA  OTEE 10  LOG  Y. 

surface  of  the  gelatin.  They  do  not  cause  liquefaction 
of  the  gelatin. 

If  grown  in  slant-  or  stab-cultures,  the  surface-devel- 
opment is  very  limited ;  along  the  needle-track  tiny 
whitish  or  bluish-white  granules  appear. 

On  nutrient  agar-agar  the  colonies  are  almost  trans- 
parent, more  or  less  glistening,  and  very  delicate  in 
structure.  On  blood-serum  development  is  more 
marked,  though  still  extremely  feeble,  appearing  as 
a  cluster  of  isolated  fine  points  growing  closely  side  by 
side. 

Growth  on  potato  is  not  usually  observed. 

When  grown  in  milk  it  commonly  causes  an  acid 
reaction  with  coincident  coagulation  of  the  casein. 
Some  varieties,  especially  non-virulent  ones,  do  not 
coagulate  milk.1 

It  is  not  motile. 

It  grows  best  at  a  temperature  of  from  35°  to  38°  C. 
Below  24°  C.  there  is  usually  no  development,  but  in  a 
few  cases  it  has  been  seen  to  grow  at  as  low  a  tempera- 
ture as  18°  C.  Above  42°  C.  development  is  checked. 

It  grows  as  well  without  as  with  oxygen.  It  is 
therefore  one  of  the  facultative  anaerobic  forms. 

Cultivation  of  this  organism  is  most  successful  when 
the  agar-agar-gelatin  mixture  of  Guarniari  is  employed. 
(See  this  medium.) 

It  may  be  stained  with  the  ordinary  aniline  staining- 
reagents.  For  demonstrating  the  capsule  the  method 
of  Gram  and  the  acetic-acid  method  give  the  best  re- 
sults. (See  Stainings.) 

This  organism  is  conspicuous  for  the  irregularity  of 
its  behavior  when  grown  under  artificial  conditions : 

i  Welch :  loc.  cit. 


BACTERIUM  PNFJUMONIJS.  335 

usually  it  loses  its  pathogenic  properties  after  a  few 
generations ;  but  again  this  peculiarity  may  be  retained 
for  a  much  longer  time.  Not  rarely  it  fails  to  grow 
after  three  or  four  transplantations  on  artificial  media, 
though  at  times  it  may  be  carried  through  many  gen- 
erations. 

INOCULATION  INTO  ANIMALS. — The  results  of  inocu- 
lations with  pure  cultures  of  this  organism  are  also  con- 
spicuous for  their  irregularity.  When  the  organism  is 
of  full  virulence  the  form  of  septicaemia  just  described 
is  usually  produced,  but  at  times  it  is  found  to  be  totally 
devoid  of  pathogenic  powers :  between  these  extremes 
cultures  may  be  obtained  possessing  every  variation  in 
the  intensity  of  their  disease-producing  properties. 
The  principal  pathological  conditions  that  may  be  pro- 
duced by  the  inoculation  of  susceptible  animals  with 
this  organism  are,  according  to  the  degree  of  its  viru- 
lence, acute  septicaemia,  spreading  inflammatory  exuda- 
tions, and  circumscribed  abscesses.  All  three  of  these 
conditions  may  sometimes  be  produced  by  inoculating 
rabbits  with  the  same  cultures  in  varying  amounts. 

Rabbits,  mice,  guinea-pigs,  dogs,  rats,  cats,  and  sheep 
are  susceptible  to  infection  by  this  organism.  Chickens 
and  pigeons  are  insusceptible.  Young  animals,  as  a 
rule,  are  more  easily  infected  than  old  ones.  Rabbits 
and  mice  are  the  most  susceptible  of  the  animals  used 
for  experimental  purposes,  and  in  testing  the  virulence 
of  a  culture  it  is  well  to  inoculate  one  of  each,  for  the 
same  culture  may  sometimes  be  virulent  for  mice  and 
not  for  rabbits,  and  vice  versa. 

If  the  culture  is  virulent,  intravascular  or  intra- 
peritoneal  injections  into  rabbits  may  produce  rapid  and 
fatal  septicaemia ;  while  subcutaneous  inoculation  of  the 


336  BACTERIOLOGY. 

same  material  may  result  in  only  a  localized  inflamma- 
tory process.  On  the  other  hand,  subcutaneous  inocula- 
tion of  less  virulent  cultures  may  produce  a  local  process, 
while  intravenous  inoculation  may  be  without  result. 
This  organism  is  the  cause  of  a  number  of  pathological 
conditions  in  human  beings  that  have  not  hitherto  been 
considered  as  related  to  one  another  etiologically.  It 
is  always  present  in  the  inflamed  area  of  the  lung  in 
acute  fibrinous  or  lobar  pneumonia  ;  it  is  known  to  cause 
acute  cerebro-spinal  meningitis,  endo-  and  peri-carditis, 
certain  forms  of  pleuritis,  arthritis  and  peri-arthritis, 
and  otitis  media. 

ANTIPNEUMONIC  SERUM. — The  recent  experiments 
of  Panichi1  on  the  serum  therapy  of  pneumonia  are  of 
great  interest.  In  order  to  obtain  a  high  grade  of  anti- 
pneumonic  serum,  he  first  perfected  a  special  culture 
medium  (a  special  bouillon)  in  which  the  organisms 
produced  their  specific  toxin  outside  the  body.  Cultures 
grown  in  this  special  medium  killed  rabbits  acutely  with 
moderate  toxic  action,  while  ordinary  blood-cultures 
killed  by  inducing  septicaemia. 

In  testing  the  curative  properties  of  his  antipneumonic 
serum,  Panichi  injected  rabbits  subcutaneously  with  0.2 
c.c.  of  his  virulent  culture,  and  subsequently  a  dose  of 
serum  was  injected  into  the  ear  veins,  in  proportion  to 
the  body-weight  of  the  animal.  A  dose  of  0.25  per  cent, 
of  the  body-weight  sufficed  to  save  the  animal  if  adminis- 
tered not  later  than  the  elapse  of  five-sixths  of  the  dura- 
tion of  the  disease,  while  the  control-animals  died  in 
from  twenty  to  fifty-six  hours.  If  the  curative  dose  of 
serum  is  delayed  longer,  2  per  cent,  of  the  body-weight 
is  required  instead  of  only  0.25  per  cent.  Panichi  con- 

1  Panichi:  Cent.  f.  Bact.,  Bd.  xxxv.,  Eeferat. 


SARCINA   TETRAGENA.  337 

eludes  that  the  serum  does  not  possess  any  bactericidal 
power,  but  that  it  acts  through  its  antitoxic  properties. 

Panichi  employed  his  serum  in  the  treatment  of  seven 
cases  of  pneumonia.  The  serum  was  injected  intrave- 
nously, and  was  followed  by  lysis — that  is,  there  was  a 
reduction  of  the  temperature,  of  the  pulse,  and  of  the 
respiration,  as  well  as  improvement  in  the  general  ap- 
pearance of  the  patient.  The  bright  red  exudate  be- 
came rusty  and,  later,  catarrhal  in  character.  Diminished 
crepitation  was  noticed  when  a  sufficient  dose  had  been 
reached  (15  to  30  c.c.).  Of  the  seven  cases,  six  expe- 
rienced a  helpful  action.  In  one  the  result  was  negative. 
In  the  latter  the  treatment  was  undertaken  at  five- 
sevenths  of  the  total  duration  of  the  disease.  The  treat- 
ment of  human  beings  corresponded  entirely  with  the 
results  of  the  experimental  study  of  the  serum  in  ani- 
mals, in  the  quantity  of  serum  required  for  treatment,  as 
well  as  in  the  time  period  within  which  the  injection  was 
of  value. 

INFECTION     WITH     SARCINA     TETRAGENA     (GAFFKY), 
MIGULA,    1900. 

Synonym  :  M icrococcus  tetragenus,  Gaffky,  1883. 

Should  the  death  of  the  animal  not  occur  within  the 
first  twenty-eight  to  thirty  hours  after  inoculation,  but 
be  postponed  until  between  the  fourth  and  eighth  day,  it 
may  result  from  the  invasion  of  the  tissues  by  the  organ- 
ism now  to  be  described,  viz.,  sarcina  tetragena. 

This  organism  was  discovered  by  Gaffky,  and  was 

subsequently  described  by  Koch  in  the  account  of  his 

experiments  upon  tuberculosis.     It  is  often  present  in 

the   saliva   of   healthy    individuals   and   is   commonly 

22 


338  B  A  CTERIOL  OGY. 

present  in  the  sputum  of  tuberculous  patients.  Koch 
found  it  very  frequently  in  the  pulmonary  cavities  of 
phthisical  patients.  It,  however,  plays  no  part  in  the 
etiology  of  tuberculosis. 

It  is  a  small  round  coccus  of  about  1  /jt  transverse 
diameter.  It  is  seen  as  single  cells,  joined  in  pairs, 
and  in  threes ;  but  its  most  conspicuous  grouping  is  in 
fours,  from  which  arrangement  it  takes  its  name.  In 
preparations  made  from  cultures  of  this  organism  it 
is  not  rare  to  find,  here  and  there,  single  bodies  which 
are  much  larger  than  the  other  individuals  in  the  field. 
Close  inspection  reveals  them  to  be  cells  in  the  initial 
stage  of  division  into  twos  and  fours.  A  peculiarity 
of  this  organism  is  that  the  cells  are  bound  together 
by  a  transparent  gelatinous  mass. 

When  cultivated  artificially  it  grows  very  slowly. 

Upon  gelatin  plates  the  colonies  appear  as  round, 
sharply  circumscribed,  punctiform  masses  which  are 
slightly  elevated  above  the  surface  of  the  surrounding 
medium.  Under  a  low  magnifying  power  they  are  seen 
to  be  slightly  granular  and  to  present  a  more  or  less 
glassy  lustre. 

The  colonies  increase  but  little  in  size  after  the  third 
or  fourth  day.  If  cultivated  as  stab-cultures  in  gelatin, 
there  appears  upon  the  surface  at  the  point  of  inocula- 
tion a  circumscribed  white  point,  slightly  elevated  above 
the  surface  and  limited  to  the  immediate  neighborhood 
of  the  point  of  inoculation.  Down  the  needle-track  the 
growth  is  not  continuous,  but  appears  in  isolated,  round, 
dense  white  clumps  or  beads,  which  do  not  develop 
beyond  very  small  points. 

It  does  not  liquefy  gelatin. 

Upon  plates  of  nutrient  agar-agar  the  colonies  appear 


SARCTNA    TETRAGENA.  339 

as  small,  almost  transparent,  round  points,  which  have 
about  the  same  color  and  appearance  as  a  drop  of  egg- 
albumin ;  they  are  very  slightly  opaque.  They  are 
moist  and  glistening.  They  rarely  develop  to  an  extent 
exceeding  1  to  2  mm.  in  diameter. 

Upon  agar-agar  as  stab-  or  slant-cultures  the  surface- 
growth  has  more  or  less  of  a  mucoid  appearance.  It 
is  moist,  glistening,  and  irregularly  outlined.  The  out- 
line of  the  growth  depends  upon  the  moisture  of  the 
agar-agar.  It  is  slightly  elevated  above  the  surface  of 
the  medium. 

In  contradistinction  to  the  gelatin  stab-cultures,  the 
growth  in  agar-agar  is  continuous  along  the  track  of 
the  needle. 

The  growth  on  potato  is  a  thick,  irregular,  slimy- 
looking  patch. 

The  transparent  mucilaginous  substance  which  is  seen 
to  surround  these  organisms  renders  them  coherent,  so 
that  efforts  to  take  up  a  portion  of  a  colony  from  the 
agar-agar  or  potato  cultures  result  usually  in  drawing 
out  fine,  silky  threads,  consisting  of  organisms  imbedded 
in  the  mucoid  material. 

The  organism  grows  best  at  from  35°  to  38°  C.,  but 
can  be  cultivated  at  the  ordinary  room-temperature — 
about  20°  C. 

The  growth  under  all  conditions  is  slow. 

It  grows  both  in  the  presence  of  and  without  oxygen. 

It  is  not  motile. 

It  stains  readily  with  all  the  ordinary  aniline  dyes. 

In  tissues  its  presence  is  readily  demonstrated  by  the 
staining-method  of  Gram. 

The  grouping  into  fours  is  particularly  well  seen  in 
sections  from  the  organs  of  animals  dead  of  this  form 


340  BACTP:RIOLOGY. 

of  septicaemia.  In  such  sections  the  organisms  will 
always  be  found  within  the  capillaries. 

INOCULATION  INTO  ANIMALS. — To  the    naked  eye 

no  alteration  can  be  seen  in  the  organs  of  animals  that 

O 

have  died  as  a  result  of  inoculation  with  sarcina 
tetragena ;  but  microscopic  examination  of  cover-slip 
preparations  from  the  blood  and  viscera  reveals  the 
presence  of  the  organisms  throughout  the  body — espe- 
cially is  this  true  of  preparations  from  the  spleen. 
White  mice  and  guinea-pigs  are  susceptible  to  the  dis- 
ease. Gray  mice,  dogs,  and  rabbits  are  not  susceptible 
to  this  form  of  septicaemia.  Subsequent  inoculation  of 
healthy  animals  with  a  drop  of  blood,  a  bit  of  tissue,  or 
a  portion  of  a  pure  culture  of  this  organism  from  the 
body  of  an  animal  dead  of  this  disease,  results  in  a  re- 
production of  the  conditions  found  in  the  dead  animal 
from  which  the  tissues  or  cultures  were  obtained. 

It  sometimes  happens  that  in  guinea-pigs  which  have 
been  inoculated  with  this  organism  local  pus-formations 
result,  instead  of  a  general  septicaemia.  The  organisms 
will  then  be  found  in  the  pus-cavity. 

BACTERIUM    INFLUENZA   (R.   PFEIFFER)   LEHMANN 

AND   NEUMANN,  1896. 

Synonym:  Influenza  bacillus,  R.  Pfeiffer,  1892. 

An  important  historic  epidemic  disease,  on  the  nature 
of  which  much  light  has  been  shed  through  modern 
methods  of  investigation,  is  influenza.  Quoting  Hirsch, 
the  first  trustworthy  literary  records  that  we  have  of 
this  disease  date  from  the  early  part  of  the  twelfth 
century. 

Between  1173  and  1874  it  made  its  epidemic  or  pan- 


BACTERIUM  INFLUENZA.  341 

clemic  appearance  on  eighty-six  different  occasions.  Its 
first  appearance  in  this  country  was  in  Massachusetts  in 
1627;  since  that  time  there  have  been  twenty -two  vis- 
itations of  influenza  to  the  United  States.  The  pan- 
demic of  1889— '90,  the  most  severe  for  a  long  time, 
appears  to  have  originated  in  Central  Asia  and  to  have 
spread  pretty  much  over  the  entire  civilized  world.  The 
advent  of  influenza  in  a  community  is  always  remark- 
able for  its  astonishing  rate  of  transmission  from  per- 
son to  person  and  its  dissemination  over  wide  areas. 

During  the  recent  pandemic  •  investigations  having 
for  their  object  the  discovery  of  its  cause  were  insti- 
tuted, with  the  result  of  demonstrating  in  the  catarrhal 
secretions  from  the  air-passages  a  micro-organism  that 
is  claimed  to  stand  in  causal  relation  to  influenza. 

Auerbach  l  examined  over  700  cultures  prepared  from 
cases  of  diphtheria  and  scarlet  fever  for  the  presence 
of  the  influenza  bacillus,  and  had  38  positive  results,  of 
which  12  were  from  cases  of  diphtheria,  3  from  scarlet 
fever,  6  from  diplitheria  and  scarlet  fever,  7  from  diph- 
theria and  measles,  and  10  from  suspicious  diphtheria 
angina.  He  employed  the  culture,  and  also  the  staining, 
methods  on  pigeon-blood  agar,  the  latter  consisting  in 
the  staining  of  the  preparations  with  the  Gram-Weigert 
stain,  followed  with  a  diluted  carbol-glycerine-fuchsin 
stain.  By  this  staining  method  it  was  not  at  all  difficult 
to  detect  the  presence  of  bacterium  influenza?.  It  ap- 
peared as  thin  reddish-violet  rods  in  contrast  to  the  dark 
blue  diphtheria  organisms  and  streptococci  and  pneu- 
mococci. 

By  appropriate  methods  of  staining  it  is  also  fre- 
quently possible  to  demonstrate  the  presence  of  this 

1  Auerbach :  Zeitsclir.  f.  Hygiene,  Bd.  47,  1904,  p.  259. 


342  BA  CTKRIOLOG  Y. 

organism  in  the  secretions  of  the  nose,  mouth,  and 
throat  of  apparently  healthy  persons. 

This  organism,  bacillus  influenza,  as  it  is  called,  was 
discovered,  isolated,  cultivated,  and  described  by  R. 
Pfeiffer. 

It  is  a  very  small,  slender,  non-spore-forming,  non- 

FlG.  04. 


^ 

X 


Bacterium  influenzse  in  sputum. 

motile,  aerobic  bacillus,  occurring  singly  and  in  pairs, 
joined  end  to  end.  It  stains  with  watery  solutions  of 
the  ordinary  basic  aniline  dyes ;  somewhat  better  with 
alkaline  methylene-blue,  but  best  when  treated  for  five 
minutes  with  a  dilution  of  Ziehl's  carbol-fuchsin  in 
water  (the  color  of  the  solution  should  be  pale  red). 
(Fig.  64.)  It  is  decolorized  by  the  method  of  Gram. 

It  develops  only  at  temperatures  ranging  from  26° 
to  43°  C.  Its  optimum  temperature  for  growth  is 
37°  C.  It  possesses  the  peculiarity  of  developing  upon 
onlv  those  artificial  culture-media  to  which  blood  or 


BACTERIUM  INFLUENZAS.  343 

blood-coloring-matter  has  been  added.  Its  cultivation 
is  best  conducted  and  its  development  most  satisfac- 
torily observed  by  the  following  procedure :  over  the 
surface  of  a  slanted  agar  tube  or  over  agar-agar  solid- 
ified in  a  Petri  dish  smear  a  small  quantity  of  sterile 
blood  (not  blood-serum).  A  bit  of  the  mucus  from  the 
sputum  of  the  influenza  patient  is  then  taken  up  with 
sterilized  forceps  or  on  a  sterilized  wire  loop,  rinsed 
in  sterile  bouillon  or  water  and  rubbed  over  the  sur- 
face of  the  prepared  agar-agar.  The  plate  or  tube  is 
then  placed  in  the  incubator  at  37°  to  38°  C.  If  in- 
fluenza bacilli  be  present,  they  will  develop  as  minute, 
transparent,  watery  colonies  that  are  without  structure, 
and  which  resemble  somewhat  minute  drops  of  dew. 
They  are  discrete  and  show  little  or  no  tendency  to 
coalesce. 

If  a  small  bit  of  mucus  be  rubbed  over  the  surface 
of  ordinary  nutrient  agar-agar,  no  such  colonies  de- 
velop. In  making  the  diagnosis  by  this  method  cult- 
ures on  both  agar-agar  containing  blood  (not  blood- 
serum)  and  agar-agar  containing  no  blood  should  always 
be  made,  for  the  reason  that  growth  of  these  peculiar 
colonies  in  the  former  and  no  such  growth  in  the  latter 
are  evidence  that  one  is  dealing  with  materials  from  a 
case  of  influenza. 

The  organism  may  also  be  cultivated  in  bouillon  to 
which  blood  has  been  added,  if  kept  at  body-tempera- 
ture. The  growth  appears  as  whitish  flakes.  Since  this 
organism  is  a  strict  aerobe,  its  cultivation  can  only  be 
conducted  on  the  surface  of  the  medium  used — i.  e., 
where  it  has  freest  access  to  oxygen.  It  is  therefore 
inadvisable  to  prepare  plates  in  the  usual  way.  When 
its  cultivation  is  attempted  in  bouillon  it  is  recom- 


344  BA  CTERIOL  OGY. 

mended,  in  order  to  favor  the  free  diffusion  of  oxygen, 
that  the  depth  of  fluid  be  very  shallow. 

Contrary  to  what  might  be  supposed,  bacterium 
influenza  has  very  little  tenacity  of  life  outside  of  the 
diseased  body.  It  is  destroyed  in  from  two  to  three 
hours  by  rapid  drying,  and  in  from  eight  to  twenty- 
four  hours  when  dried  more  slowly.  Cultures  retain 
their  vitality  for  from  two  to  three  weeks.  The  organ- 
ism dies  in  water  in  a  little  over  a  day.  As  a  result  of 
these  observations,  Pfeiffer  does  not  believe  the  disease 
to  be  disseminated  by  either  the  air  or  the  water,  but 
rather  by  direct  infection  from  the  catarrhal  secretions 
of  the  patients. 

This  organism  has  not  been  found  outside  of  the 
human  body.  In  the  influenza  patient  it  is  present  in 
the  catarrhal  secretions,  bronchial  mucous  membrane, 
and  the  diseased  lung-tissues.  It  may  be  demonstrated 
microscopically  in  the  mucus  by  cover-slip  prepara- 
tions made  in  the  usual  way  and  stained  with  diluted 
carbol-fuchsin,  referred  to  above.  In  the  tissues  it 
may  be  demonstrated  in  sections  stained  in  the  same 
solution.  In  the  sputum  the  bacteria  are  found  as 
masses  and  as  scattered  cells.  (See  Fig.  64.)  They  are 
also  found  within  the  bodies  of  leucocytes,  especially  in 
the  later  stages  of  the  disease  when  convalescence  has 
set  in ;  at  this  time  they  appear  as  very  small,  irregular, 
evidently  degenerated  bacteria  within  white  blood- 
corpuscles.  They  are  also  present  in  the  nasal  secre- 
tions. 

At  autopsies  it  is  advisable  to  cut  out  pieces  of 
the  diseased  tissue  about  the  size  of  a  pea  or  a  bean, 
break  them  up  in  a  small  quantity  of  sterile  water  or 
bouillon,  and  make  the  cultures  from  this  infusion. 


BACTERIUM  INFLUENZA.  345 

By  this  procedure  two  advantages  are  gained :  first, 
a  dilution  of  the  number  of  bacteria  present;  and, 
secondly,  the  tissue  furnishes  the  amount  of  haemo- 
globin necessary  for  the  growth  of  the  organism. 
Under  these  circumstances  it  is,  of  course,  not  neces- 
sary to  make  a  further  addition  of  blood  to  the  culture- 
medium. 

The  only  animal  that  has  been  found  susceptible 
to  inoculation  with  this  organism  is  the  monkey.  By 
intratracheal  injection  PfeifFer  succeeded  in  causing  a 
toxic  condition  that  proved  fatal.  He  does  not  regard 
the  death  of  the  animals  as  due  to  infection,  but  rather 
to  intoxication.  The  disease,  as  seen  in  man,  has  not 
been  reproduced  in  animals. 


CHAPTER    XVII. 

Tuberculosis — Microscopic  appearance  of  miliary  tubercles — Diffuse 
caseation — Cavity-formation — Encapsulation  of  tuberculous  foci — 
Primary  infection — Modes  of  infection — Location  of  the  bacilli  in 
the  tissues — Staining-peculiarities — Organisms  with  which  bac- 
terium tuberculosis  may  be  confounded:  bacterium  leprte  ;  bacterium 
smegmatis — Points  of  differentiation — Acid-proof  bacteria — Actino- 
myces — Actinomyces  bovis,  Actinomyces  Israeli,  Actinomyces  madurte, 
Actinomyces  farcinicus,  Actinomyces  Eppingeri,  Actinomyces  pseudo- 
tuberculosis. 

BACTERIUM    TUBERCULOSIS    (KOCH),  MIGULA,  1900. 
Synonym  :  Bacillus  tuberculosis,  Koch,  1882. 

LOCAL  OR  GENERAL  TUBERCULOSIS. — Should  the 
animal  succumb  to  neither  of  the  septic  processes  just 
described,  then  its  death  from  tuberculosis  may  reason- 
ably be  expected. 

When  this  disease  is  in  progress  alterations  in  the 
lymphatic  glands  nearest  the  site  of  inoculation  may 
be  detected  by  the  touch  in  from  two  to  four  weeks. 
They  will  then  be  found  enlarged.  Though  not  con- 
stant, tumefaction  and  subsequent  ulceration  at  the 
point  of  inoculation  may  be  observed.  Progressive 
emaciation,  loss  of  appetite,  and  difficulty  in  respiration 
point  to  the  existence  of  the  general  tuberculous  process. 
Death  ensues  in  from  four  to  eight  weeks  after  inocula- 
tion. At  autopsy  either  general  or  local  tuberculosis 
may  be  found.  The  expressions  of  tuberculosis  are  so 
manifold  and  in  different  animals  vary  so  widely  the 
one  from  the  other,  that  no  fixed  law  as  to  what  will 
appear  at  autopsy  can  ct  priori  be  laid  down. 

The  guinea-pig,  which  is  best  suited  for  this  experi- 
ment because  its  susceptibility  to  tuberculosis  is  greater 
and  more  constant  than  that  of  other  animals  usually 

346 


LOCAL   OR   GENERAL   TUBERCULOSIS.          347 

found  in  the  laboratory,  presents,  in  the  main,  changes 
that  are  characterized  by  coagulation-necrosis  and  case- 
ation.  This  is  particularly  the  case  when  the  infec- 
tion is  general — i.  e.,  when  the  process  is  of  the  acute 
miliary  type ;  then  the  tissues  of  the  liver  and  spleen 
present  the  most  favorable  field  for  the  study  of  this 
pathological-anatomical  alteration. 

In  general,  the  tubercular  lesions  can  be  divided  into 
those  of  strictly  focal  character — i.  e.,  the  miliary  and 
the  conglomerate  tubercles — and  those  which  are  more 
diffuse.  The  latter  lesions,  although  fundamentally 
of  the  same  nature  as  the  miliary  tubercles,  are  much 
greater  in  extent  and  not  so  sharply  circumscribed. 
These  latter  lesions  play  a  more  conspicuous  role  in  the 
pathology  of  the  disease  than  do  the  miliary  nodules, 
although  it  is  to  the  presence  of  the  miliary  nodules  that 
the  disease  owes  its  name. 

At  autopsy  the  pathological  manifestations  of  the  dis- 
ease are  not  infrequently  seen  to  be  confined  to  the  seat 
of  inoculation  and  to  the  neighboring  lymphatic  glands. 
These  tissues  then  present  all  the  characteristics  of  the 
tuberculous  process  in  the  stage  of  cheesy  degeneration. 
When  the  disease  is  more  general  the  degree  of  its  exten- 
sion varies.  Sometimes  the  small  gray  nodules — mili- 
ary tubercles — are  only  to  be  seen  with  the  naked  eye  in 
the  tissues  of  the  liver  and  spleen.  Again,  they  may  in- 
vade the  lung,  and  frequently  they  are  distributed  over 
the  serous  membranes  of  the  intestines,  the  lungs,  the 
heart,  and  the  brain.  These  gray  nodules,  as  seen  by 
the  naked  eye,  vary  in  size  from  that  of  a  pin-point  to 
that  of  a  hempseed,  and,  as  a  rule,  are,  in  this  stage,  the 
result  of  the  fusion  of  two  or  more  smaller  miliary  foci. 
Though  the  two  terms  "miliary"  and  "conglomerate" 


348  SA  CTERTOLOG  Y. 

are  employed  for  the  description  of  the  microscopic 
appearance  of  these  nodules,  yet  it  is  very  rarely  that 
any  condition  other  than  that  due  to  the  fusion  of 
several  of  these  minute  foci  can  be  detected  by  the 
naked  eye. 

The  miliary  tubercles  are  of  a  pale  gray  color,  with  a 
white  centre,  are  slightly  elevated  above  the  surface  of 
the  tissue  in  which  they  are  located,  and,  as  stated,  vary 
considerably  in  dimensions,  usually  appearing  as  points 
which  range  in  size  from  that  of  a  pin-point  to  that  of 
a  pin-head.  They  are  not  only  located  upon  the  surface 
of  the  organs,  but  are  distributed  through  the  depths  of 
the  tissues.  To  the  touch  they  sometimes  present  noth- 
ing characteristic,  but  when  closely  packed  together  in 
large  numbers  they  usually  give  a  mealy  or  sandy  sen- 
sation to  the  hand  passed  over  them.  Stained  sections 
of  miliary  tubercles  present  a  distinctly  characteristic 
appearance,  and  the  disease  may  be  diagnosticated  by 
these  histological  changes  alone,  though  the  crucial  test 
in  the  diagnosis  is  the  demonstration  of  tubercle  bacilli 
within  the  nodules. 

MICROSCOPIC  APPEARANCE  OF  MILIARY  TUBER- 
CLES.— A  miliary  tubercle  under  a  low  magnifying 
power  of  the  microscope  presents  somewhat  the  follow- 
ing appearance :  there  is  a  central  pale  area,  evidently 
composed  of  necrotic  tissue  because  of  its  incapacity 
for  taking  up  the  nuclear  stains  commonly  employed. 
Scattered  through  this  necrotic  area  may  be  seen  granular 
masses  irregular  in  size  and  shape  ;  they  take  up  the  stains 
employed  and  are  evidently  fragments  of  cell-nuclei  in 
course  of  destruction.  Through  the  necrotic  area  may 
here  and  there  be  seen  irregular  lines,  bands,  or  ridges,  the 
remains  of  tissues  not  yet  completely  destroyed  by  the 


LOCAL   OR   GENERAL   TUBERCULOSIS.          349 

necrotic  process.  Around  the  periphery  of  this  area 
may  sometimes  be  noticed  large  multinucleated  cells, 
the  nuclei  of  which  are  arranged  about  the  periphery 
of  the  cell  or  grouped  irregularly  at  its  poles.  The 
arrangement  of  these  nuclei  as  observed  in  sections 
is  usually  oval,  or  somewhat  crescentic.  In  tuber- 
cles from  the  human  subject  these  large  "  giant-cells," 
as  they  are  called,  are  quite  common.  They  are  much 
less  frequent  in  tubercular  tissues  from  lower  ani- 
mals. 

Round  about  the  central  focus  of  necrosis  is  seen  a 
more  or  less  broad  zone  of  closely  packed  small  round 
and  oval  bodies,  which  stain  readily  but  not  homoge- 
neously. They  vary  in  size  and  shape,  and  are  seen  to 
be  imbedded  in  a  delicate  network  of  fibrinous-looking 
tissue.  This  fibrin-like  network  in  which  these  bodies  lie, 
and  which  is  a  common  accompaniment  of  giant-cell  for- 
mation, is  in  part  composed  of  fibrin,  but  is  in  the  main, 
most  probably,  the  remains  of  the  interstitial  fibrous 
tissue  of  the  part.  This  zone  of  which  we  are  speak- 
ing is  the  zone  of  so-called  "granulation-tissue,"  and 
consists  of  leucocytes,  granulation-cells,  fibrin,  and  the 
fibrous  remains  of  the  organ ;  the  irregularly  oval,  gran- 
ular bodies  which  take  up  the  stain  are  the  nuclei  of 
these  cells.  The  zone  of  granulation-tissue  surrounds 
the  whole  of  the  tuberculous  process,  and  at  its  periphery 
fades  gradually  into  the  healthy  surrounding  tissues  or 
fuses  with  a  similar  zone  surrounding  another  tubercu- 
lar focus.  This  may  be  taken  as  a  description  of  the 
typical  miliary  tubercle. 

DIFFUSE  CASSATION. — The  diifuse  caseation,  as  said, 
plays  a  more  important  role  in  the  tuberculous  lesion, 
both  in  the  human  and  experimental  forms,  than  does 


350  BACTERIOLOGY. 

the  formation  of  miliary  tubercles.  In  this  a  large 
area  of  tissue  undergoes  the  same  process  of  necrosis 
and  caseation  as  the  centre  of  the  miliary  tubercle. 
In  certain  tissues,  notably  the  lungs  and  lymphatics, 
it  is  more  marked  than  in  others.  In  rabbits,  par- 
ticularly, all  the  changes  in  the  lung  frequently  come 
under  this  head.  When  this  is  the  case  solid  masses 
are  found,  sometimes  as  large  as  a  pea.  or  involving 
even  an  entire  lobe  or  the  whole  lung  in  some  cases. 
They  are  of  a  whitish-yellow,  opaque  color,  and  on  sec- 
tion are  peculiarly  dry  and  hard.  Entire  lymphatic 
glands  may  be  changed  in  this  way.  The  conditions 
which  appear  to  be  most  favorable  to  the  occurrence 
of  this  widespread  caseation  of  the  tissues  are  the 
simultaneous  deposition  of  a  large  number  of  tubercle 
bacilli  in  them,  and  the  involvement  of  a  wide  area  in- 
stead of  a  single  isolated  point,  as  in  the  miliary  tubercle. 
Necrosis  is  so  rapid  that  time  does  not  suffice  for  those 
reactive  changes  to  take  place  in  the  tissues  which  result 
in  the  formation  of  the  outer  zone  of  the  miliary  tubercle. 
In  other  instances  the  entire  caseous  area  is  surrounded 
by  a  granulation-zone  similar  to  that  around  the  caseous 
centre  of  the  miliary  tubercles.  It  is  of  special  im- 
portance to  recognize  the  etiological  connection  between 
this  diffuse  caseation  and  the  tubercle  bacillus,  because 
until  its  nature  was  accurately  determined  caseous  pneu- 
monia of  the  lungs  formed  the  chief  obstacle  which 
many  encountered  in  recognizing  the  specific  infectious- 
ness  of  tuberculosis. 

CAVITY-FORMATION. — The  production  of  cavities,  a 
prominent  feature  in  human  tuberculosis,  particularly 
of  the  lungs,  is  due  to  softening  of  the  necrotic, 
caseous  masses  or  of  aggregations  of  miliary  tuber- 


LOCAL  OR  GENERAL  TUBERCULOSIS.         351 

cles.  The  material  softens  and  is  expelled,  and  a 
cavity  remains.  In  the  wall  of  this  cavity  the  tuber- 
culous changes  still  proceed,  both  as  diffuse  caseation 
and  formation  of  miliary  tubercles.  The  whole  cavity 
with  the  reactive  changes  in  the  tissues  of  its  walls 
may  be  properly  conceived  as  a  single  gigantic  tuber- 
cle, its  Avail  forming  a  tissue  very  analogous  to  the  outer 
zone  of  the  single  tubercle,  the  cavity  itself  correspond- 
ing to  the  caseous  centre. 

In  animals  used  for  experiment  cavity-formation  of 
this  sort  is  very  rare,  owing  to  the  greater  resistance  of 
the  caseous  tissue.  That  it  is,  however,  possible  to 
produce  in  rabbits  pulmonary  cavities  in  all  physical 
respects  similar  to  those  seen  in  the  human  being 
has  been  beautifully  demonstrated  by  Prudden.  He 
showed  that  when  he  had  injected  fluid  cultures  of  strep- 
tococcus pyogenes  into  the  trachea  of  rabbits  already  af- 
fected with  tubercular  consolidation  of  the  lungs,  the 
result  of  the  mixed  infection  thus  brought  about  was 
cavity-formation  in  eight  out  of  nine  lungs  subjected  to 
the  conditions  of  the  experiment ;  while  in  only  one  out 
of  eleven  did  cavities  form  under  the  influence  of  the 
tubercle  bacillus  alone.1 

In  the  contents  and  in  the  walls  of  tubercular  cavi- 
ties in  man  bacteria  other  than  bacillus  tuberculosis  are 
found.  It  is  to  the  influence  of  some  of  these,  as  we 
have  seen,  that  diseases  other  than  tuberculosis  may 
sometimes  be  produced  by  the  inoculation  of  animals 
with  the  sputum  from  such  cases;  and  it  is  also  to  the 
absorption  of  their  toxic  products  that  some  of  the  con- 

1  Prudden  :  "  Experimental  Phthisis  in  Rabbits,  with  the  Formation 
of  Cavities/'  etc.,  Transactions  of  the  Association  of  American  Physi- 
cians, 1894,  vol.  ix.  p.  166. 


352  BA  CTERIOL  OGY. 

stitutional  manifestations  commonly  seen  in  cases  of 
advanced  pulmonary  tuberculosis  are  attributed. 

ENCAPSULATION  OF  TUBERCULAR  Foci. — It  not 
uncommonly  occurs  that  round  about  a  necrotic  tuber- 
culous focus  there  is  formed  a  fibrous  capsule  which  may 
completely  shut  off  the  diseased  from  the  healthy  tissue 
surrounding  it ;  or  a  tuberculous  focus  may,  through  the 
resistance  of  the  tissue  in  which  it  is  located,  be  more 
or  less  completely  isolated.  In  this  condition  the  dis- 
eased foci  may  lie  dormant  for  a  long  time  and  give  no 
evidence  of  their  existence,  until  they  are  caused  to 
break  through  their  envelopes  by  some  disturbing 
cause.  With  the  passage  of  the  bacilli  or  their  spores 
from  such  a  focus  into  the  vascular  or  lymphatic  cir- 
culation the  disease  may  become  general. 

It  is  to  some  such  accident  as  this  that  the  sudden 
appearance  of  general  tubercular  infection  in  subjects 
supposed  to  have  recovered  from  the  primary  local 
manifestations  may  often  be  attributed.  The  breaking- 
down  of  old  caseous  lymphatic  glands  is  a  common 
example  of  this  recurrence  of  tuberculosis. 

PRIMARY  INFECTION.  —  Primary  infection  occurs 
through  either  the  vascular  or  lymphatic  circulation. 
Through  these  channels  the  bacilli  gain  access  to  the 
tissues  and  become  lodged  in  the  finer  capillary  ramifi- 
cations or  in  the  more  minute  lymph-spaces.  Here 
they  find  conditions  favorable  to  their  development, 
and  in  the  course  of  their  life-processes  produce  sub- 
stances of  a  chemical  nature  which  serve  to  bring 
about  characteristic  changes  in  the  immediate  neighbor- 
hood. In  the  beginning  the  fixed  cells,  particularly  the 
endothelial  cells  of  the  capillaries  and  lymph-spaces, 
are  stimulated  to  proliferation.  With  the  onset  of  this 


LOCAL   OR   GENERAL   TUBERCULOSIS         353 

phenomenon  karyokinesis,  evidence  of  cell  multiplica- 
tion may  readily  be  detected  in  and  about  the  affected 
focus.  As  proliferation  continues  and  as  the  local  cir- 
culation becomes  more  and  more  impaired,  the  centre  of 
the  diseased  area  gradually  assumes  a  condition  of  in- 
activity, and  ultimately  presents  all  the  characteristics 
of  dead  and  dying  tissue.  This  death  of  tissue  is  one 
of  the  earliest,  the  most  easily  recognized,  and  the  most 
characteristic  results  of  tubercular  infection,  and  may 
usually  be  detected,  in  greater  or  less  degree,  even  in 
the  youngest  and  most  minute  tubercles.  With  the  pro- 
duction of  this  progressive  necrosis — for  progressive  it  is, 
as  it  proceeds  as  long  as  the  bacilli  live  and -continue  to 
produce  their  poisonous  products — there  is  in  addition 
a  reactive  change  in  the  surrounding  tissues,  which 
consists  in  the  formation  of  a  granulation-zone  at  the 
outer  margins  of  the  dying  and  dead  tissue.  This  zone 
consists  of  small,  round  granulation-cells  and  of  leuco- 
cytes, all  of  which  are  seen  in  the  meshes  of  the  finer 
fibrous  tissues  of  the  part.  At  the  same  time  altera- 
tions are  produced  in  the  walls  of  the  vessels  of  the 
locality  ;  these  tend  to  occlude  them,  and  thus  the  proc- 
ess of  tissue-death  is  favored  by  a  diminution  of  the 
amount  of  nutrition  brought  to  them.  These  changes 
may  continue  until  eventually  conglomerate  tubercles, 
widespread  caseation,  or  cavity-formation  results ;  or 
from  one  cause  or  another  the  life-processes  of  the 
bacilli  may  be  checked  and  recovery  occur. 

MODES  OF  INFECTION. — Experimentally,  tuberculosis 
may  be  produced  in  susceptible  animals  by  subcutaneous 
inoculation,  by  direct  injection  into  the  circulation, 
by  injection  into  the  peritoneal  cavity,  by  feeding  of 
tuberculous  material,  by  the  introduction  of  the  bacilli 

23 


354  B  A  CTEEIOL  OGY. 

into  the  air-passages,  and  by  inoculation  into  the  ante- 
rior chamber  of  the  eye. 

In  the  human  subject  the  most  common  portals  of 
infection  are,  doubtless,  the  air-passages,  the  alimentary 
tract,  and  cutaneous  wounds.  When  introduced  subcu- 
taneously  the  resulting  process  finds  its  most  pronounced 
expression  in  the  lymphatic  system.  The  growing 
bacilli  make  their  way  into  the  lymphatic  spaces  of 
the  loose  cellular  tissue,  are  taken  up  in  the  lymph- 
stream  and  deposited  in  the  neighboring  lymphatic 
glands.  Here  they  may  remain  and  give  rise  to  no 
alteration  other  than  that  seen  in  the  glands  them- 
selves ;  or  they  may  pass  on  to  neighboring  glands, 
and  eventually  be  disseminated  throughout  the  lym- 
phatic system,  ultimately  reaching  the  vascular  system. 

Having  gained  access  to  the  bloodvessels  the  results 
are  the  same  as  those  following  intravascular  injection 
of  the  bacilli,  namely :  general  tuberculosis  quickly 
follows,  with  the  production  of  miliary  tubercles  most 
conspicuous  in  the  lungs  and  kidneys ;  less  numerous 
in  the  spleen,  liver,  and  bone-marrow. 

When  inhaled  into  the  lungs,  if  conditions  are  favor- 
able, multiplication  of  the  bacilli  quickly  follows.  Co- 
incident with  their  growth  they  are  mechanically  pressed 
into  the  tissues  of  the  lungs.  As  multiplication  con- 
tinues some  are  transported  from  the  primary  site  of 
infection  to  healthy  portions  of  the  lung-tissue,  where, 
through  their  development,  the  process  is  repeated. 

In  the  same  way  infection  by  way  of  the  alimentary 
tract  is  in  the  main  due  to  the  bacilli  being  forced  by 
mechanical  pressure  into  the  walls  of  the  intestines.  In- 
vestigation has  shown  that  lesions  of  the  intestinal  coats 
are  not  necessary  for  the  entrance  of  tubercle  bacilli 


LOCAL   OR   GENERAL   TUBERCULOSIS.         355 

from  the  lumen  of  the  intestines  into  the  internal 
organs  and  tissues.  They  may  be  transported  from 
the  intestinal  tract  into  the  lymphatics  in  the  same 
way  that  the  fat-droplets  of  the  chyle  find  entrance 
into  the  lymphatic  circulation. 

Unlike  most  pathogenic  organisms,  the  tubercle  ba- 
<'illus  is  believed  to  have  the  property  of  forming  spores 
within  the  tissues.  These  spores,  which  are  presum- 
ably highly  resistant  and  not  destroyed  by  drying, 
are  thrown  off  from  the  lungs  in  the  sputum  of  tuber- 
culous patients  in  large  numbers ;  and  unless  special 
precautions  be  taken  to  prevent  it,  the  sputum  becomes 
dried,  is  ground  into  dust,  and  sets  free  in  the  atmos- 
phere the  spores  of  tubercle  bacilli  which  came  with  it 
from  the  lungs,  and  which  have  the  property  of  ex- 
citing the  disease  in  a  certain  number  of  persons  who 
inhale  them.  The  frequency  of  pulmonary  tuber- 
culosis points  to  this  as  one  of  the  commonest  sources 
and  modes  of  infection  in  human  beings.  This  opinion 
is  borne  out  by  statistical  studies  upon  the  disease,  as 
well  as  by  such  evidence  as  Cornet  *  has  produced  upon 
the  infective  nature  of  dust  taken  from  apartments 
occupied  by  persons  suffering  from  pulmonary  tuber- 
culosis. 

LOCATION  OF  THE  BACILLI  IN  THE  TISSUES. — The 
bacilli  will  be  found  most  numerous  in  those  tissues 
which  are  the  seats  of  the  active  stage  of  the  process. 

In  the  initial  stage  of  the  disease  the  bacilli  will 
be  fewer  in  number  than  later.  At  this  time  only 
single  rods  may  here  and  there  be  found ;  later  they 
are  more  numerous ;  and,  finally,  when  the  process 
has  advanced  to  a  stage  easily  recognizable  by  the  naked 

1  Cornet:  Zeitschrift  fur  Hygiene,  1889,  Bd.  v.  S.  191. 


356  BACTERIOLOG  Y. 

eye,  they  are  found  in  the  granulation-zones  in  clumps 
and  scattered  about  in  large  numbers. 

In  the  central  necrotic  masses,  which  consist  of  cell- 
detritus,  it  is  rare  that  the  organisms  can  be  demon- 
strated microscopically.  It  is  at  the  periphery  of  these 
areas  and  in  the  progressing  granular  zone  that  they 
are  most  frequently  to  be  seen. 

This  apparent  absence  of  the  bacilli  from  the  central 
necrotic  area  and  often  from  old  caseous  tissues  must 
not  be  taken,  however,  as  evidence  that  these  materials 
are  not  infective.  As  bacilli,  they  are  difficult  to 
demonstrate  here  because  the  probabilities  are  that  at 
this  stage  of  the  process,  owing  to  conditions  unfavor- 
able to  their  further  growth,  they  are  in  the  spore- 
stage,  a  stage  in  which  it  is  as  yet  impossible,  with 
our  present  methods  of  staining,  to  render  them  visi- 
ble. The  facts  that  this  tissue  is  infective,  and  that 
with  it  the  disease  can  be  reproduced  in  susceptible 
animals,  speak  for  the  accuracy  of  this  assumption. 
A  conspicuous  example  of  this  condition  is  seen  in 
old  scrofulous  glands.  These  glands  usually  present 
a  slow  process,  are  commonly  caseous,  and  always 
possess  the  property  of  producing  the  disease  when 
introduced  into  the  tissues  of  susceptible  animals,  and 
yet  they  are  the  most  difficult  of  all  tissues  in  which  to 
demonstrate  microscopically  the  presence  of  tubercle 
bacilli. 

In  tubercles  containing  giant-cells  the  bacilli  can 
usually  be  demonstrated  in  the  granular  contents  of 
these  cells.  Frequently  they  will  be  found  accumu- 
lated at  the  pole  of  the  cell  opposite  to  that  occupied 
by  the  nuclei,  as  if  there  existed  an  antagonism  between 
the  nuclei  and  the  bacilli.  In  some  of  these  cells, 


BACTERIUM  TUBERCULOSIS.  357 

however,  the  distribution  of  the  bacilli  is  seen  to  be 
irregular,  and  they  will  be  found  scattered  among  the 
nuclei  as  well  as  in  the  necrotic  centre  of  the  cell.  As 
the  number  of  bacilli  in  the  giant-cell  increases  the  cell 
itself  is  ultimately  destroyed. 

Tubercular  tissues  always  contain  the  bacilli  or  their 
spores/  and  are  always  capable  of  reproducing  the  dis- 
ease when  introduced  into  the  body  of  a  susceptible 
animal.  From  the  tissues  of  this  animal  the  bacilli 
may  be  obtained  and  cultivated  artificially,  and  these 
cultures  are  capable  of  again  producing  the  disease 
when  further  inoculated.  Thus  the  postulates  for- 
mulated by  Koch,  which  are  necessary  to  prove  the 
etiological  role  of  an  organism  in  the  production  of  a 
malady,  are  fulfilled. 

THE    BACTERIUM    TUBERCULOSIS. 

Of  the  three  pathogenic  organisms  liable  to  occur 
in  the  sputum  of  a  tuberculous  subject,  the  tubercle 
bacillus  gives  us  the  greatest  difficulty  in  our  efforts  at 

cultivation. 

It  is,  in  the  strict  sense  of  the  word,  a  parasite,  and 
finds  conditions  entirely  favorable  to  its  development 
only  in  the  animal  body.  On  ordinary  artificial  media 
the  bacilli  taken  directly  from  the  animal  body  grow 
only  very  imperfectly,  or,  in  many  cases,  not  at  all. 
From  this  it  seems  probable  that  there  is  a  difference 
in  the  nature  of  individual  tubercle  bacilli  —  some 
appearing  to  be  capable  of  growth  in  the  animal  tis- 
sues only,  while  others  are  apparently  possessed  of  the 
power  to  lead  a  limited  saprophytic  existence.  It  may 
be,  therefore,  that  those  bacilli  which  we  obtain  as  arti- 

1  The  property  of  spore-forinatiou  is  assumed,  not  proved. 


358  BACTERIOLOGY. 

tidal  cultures  from  the  animal  body  are  offsprings  of 
the  more  saprophytic  varieties.  At  best,  one  never  sees 
with  the  tubercle  bacillus  a  saprophytic  condition  in 
any  way  comparable  to  that  possessed  by  many  of  the 
other  organisms  with  which  we  have  to  deal. 

For  the  cultivation  of  bacillus  tuberculosis  directly 
from  the  tissues  of  the  animal,  the  method  by  which  one 
obtains  the  best  results  is  that  recommended  by  Koch, 
viz.,  cultivation  upon  blood-serum.  Its  parasitic  ten- 
dencies are  so  pronounced  that  even  very  slight  variations 
in  the  conditions  under  which  one  endeavors  to  isolate 
bacillus  tuberculosis  from  the  tissues  may  cause  total 
failure.  It  is,  therefore,  necessary  that  the  injunctions 
for  obtaining  it  in  pure  culture  should  be  carefully 
observed. 

PREPARATION  OF  CULTURES  FROM  TISSUES. — Under 
strictest  antiseptic  precautions  remove  from  the  animal 
the  diseased  organ — the  liver,  spleen,  or  a  lymphatic 
gland  being  preferable.  Place  the  tissue  in  a  sterilized 
Petri  dish,  and  dissect  out  with  sterilized  scissors  and 
forceps  the  small  tubercular  nodules.  Place  each  nodule 
upon  the  surface  of  the  blood-serum,  one  nodule  in 
each  tube,  and  without  attempting  to  break  it  up  or 
smear  it  over  the  surface,  leave  it  for  four  or  five  days 
in  the  incubator.  After  this  time  it  may  be  rubbed 
over  the  surface  of  the  serum.  The  object  of  this  is  to 
give  to  the  bacilli  in  the  nodule  an  opportunity  to 
multiply,  under  the  favorable  conditions  of  temperature 
and  moisture,  before  an  effort  is  made  to  distribute  them 
over  the  surface  of  the  medium.  It  is  best  to  dissect 
away  twenty  to  thirty  such  tubercles  and  treat  each  in 
the  same  way.  Some  of  the  tubes  will  remain  sterile, 
others  may  be  contaminated  by  extraneous  saprophytic 


BACTERIUM  TUBERCULOSIS.  359 

organisms  during  the  manipulation,  while  a  few  may 
give  the  result  desired,  viz.,  a  growth  of  the  tubercle 
bacillus  itself. 

The  blood-serum  upon  which  the  organism  is  to  be 
cultivated  should  be  comparatively  freshly  prepared — 
that  is,  should  not  be  dry. 

After  inoculating  the  tubes  they  should  be  carefully 
sealed  to  prevent  evaporation  and  consequent  dry- 
ing. This  is  done  by  burning  off  the  overhanging 
cotton  plug  in  a  gas-flame,  and  then  impregnating 
the  upper  layers  of  the  cotton  with  either  sealing- 
wax  or  paraffin  of  a  high  melting-point ;  or  by  insert- 
ing over  the  burned  end  of  the  cotton  plug  a  soft, 
closely  fitting  cork  that  has  been  sterilized  in  the 
steam  sterilizer  just  before  using  (Ghriskey).  This 
precaution  is  necessary  because  of  the  slow  growth  of 
the  organism.  Under  the  most  favorable  conditions 
tubercle  bacilli  directly  from  the  animal  body  show  no 
evidence  of  growth  for  about  twelve  days  after  inocu- 
lation upon  blood-serum,  and,  as  they  must  be  retained 
during  this  time  at  the  body-temperature — 37.5°  C. — 
evaporation  would  take  place  very  rapidly  and  the 
medium  would  become  too  dry  for  their  development. 

If  these  primary  efforts  result  in  the  appearance  of  a 
culture  of  the  bacilli,  further  cultivations  may  be  made 
by  taking  up  a  bit  of  the  colony,  preferably  a  moder- 
ately large  quantity,  and  transferring  it  to  fresh  serum, 
and  this  in  turn  is  sealed  up  and  retained  at  the  same 
temperature.  Once  having  obtained  the  organism  in 
pure  culture,  its  subsequent  cultivation  may  be  con- 
ducted upon  the  glycerin-agar-agar  mixture — ordinary 
neutral  nutrient  agar-agar  to  which  from  4  to  6  per  cent, 
of  glycerin  has  been  added.  This  is  a  very  favorable 


360  BACTERIOLOGY. 

medium  for  the  growth  of  this  organism  after  it  has 
accommodated  itself  to  its  saprophytic  mode  of  exist- 
ence, though  blood-serum  is  perhaps  the  best  medium 
to  be  employed  in  obtaining  the  first  generation  of  the 
organism  from  tuberculous  tissues. 

The  organism  may  be  cultivated  also  on  neutral  milk 
to  which  1  per  cent,  of  agar-agar  has  been  added,  also 
upon  the  surface  of  potato,  and  likewise  in  meat-infu- 
sion bouillon  containing  from  4  to  6  per  cent,  of 
glycerin. 

Cultures  of  the  tubercle  bacillus  are  characteristic 
in  appearance — once  having  seen  them  there  is  little 
probability  of  subsequent  mistake.  They  appear  as 
dry  masses,  which  may  develop  upon  the  surface  of 
the  medium  either  as  flat  scales  or  as  coarse  granular 
masses.  They  are  never  moist,  and  frequently  have  the 
appearance  of  dry  meal  spread  upon  the  surface  of  the 
medium.  In  the  lower  part  of  the  tube  in  which  they 
are  growing — i.  e.,  that  part  occupied  by  a  few  drops  of 
fluid  which  has  in  part  been  squeezed  from  the  medium 
during  the  process  of  solidification,  and  is  in  part  water 
of  condensation — the  colonies  may  be  seen  to  float  as  a 
thin  pellicle  upon  the  surface  of  the  fluid. 

The  individuals  composing  the  growth  adhere  so 
tenaciously  together  that  it  is  with  the  greatest  diffi- 
culty they  can  be  separated.  In  even  the  oldest  and 
dryest  cultures  pulverization  is  impossible.  The  masses 
can  only  be  separated  and  broken  up  by  grinding  in 
a  mortar  with  the  addition  of  some  foreign  substance, 
such  as  very  fine,  sterilized  sand,  dust,  etc. 

The  cultures  are  of  a  dirty-drab  or  brownish-gray 
color  when  seen  on  serum  or  glycerin-agar-agar. 

On  potato  they  grow  in  practically  the  same-  way, 


BACTERIUM  TUBERCULOSIS.  361 

though  the  development  is  much  more  limited.  On 
this  medium  they  are  of  nearly  the  same  color  as  the 
potato  on  which  they  are  growing.  When  cultivated 
for  a  time  on  potato  they  are  said  to  lose  their  patho- 
genic properties. 

On  milk-agar-agar  they  are  of  so  nearly  the  same 
color  as  the  medium  that,  unless  they  are  growing  as 
characteristic  mealy-looking  masses,  considerably  ele- 
vated above  the  surface,  their  presence  is  less  conspicu- 
ous than  when  on  other  media. 

In  bouillon  they  grow  as  a  thin  pellicle  on  the  sur- 
face. This  may  fall  to  the  bottom  of  the  fluid  and  con- 
tinue to  develop,  its  place  on  the  surface  being  taken  by 
a  second  pellicle. 

The  tubercle  bacillus  does  not  develop  on  gelatin 
because  of  the  low  temperature  at  which  this  medium 
must  be  used. 

MICROSCOPIC  APPEARANCE  OF  BACTERIUM  TUBER- 
CULOSIS.— Microscopically  the  organism  itself  is  a 
delicate  rod,  usually  somewhat  beaded  in  its  structure, 
though  rarely  it  is  seen  to  be  homogeneous.  It  is  either 
quite  straight,  or  somewhat  curved  or  bent  on  its  long 
axis.  In  some  preparations  involution-forms,  consisting 
of  rods  a  little  clubbed  at  one  extremity  or  slightly 
bulging  at  different  points,  may  be  detected.  Branch- 
ing forms  of  this  organism  have  been  described.  It 
varies  in  length — sometimes  being  seen  in  very  short 
segments,  again  much  longer,  though  never  as  long 
tli  reads.  Usually  its  length  varies  from  2  to  5  //.  It 
is  commonly  described  as  being  in  length  about  one- 
fourth  to  one-half  the  diameter  of  a  red  blood-corpuscle. 
It  is  very  slender.  (See  Fig.  63.) 

These  rods  usually  present,  as  has  been  said,  an  ap- 


362  BACTERIOLOGY. 

pearance  of  alternate  stained  and  colorless  portions. 
The  latter  portions  are  believed  to  be  the  spores  of 
the  organism,  though  as  yet  no  incontestable  proof  of 
this  opinion  has  been  presented.  At  times  these  colorless 
portions  are  seen  to  bulge  slightly  beyond  the  contour 
of  the  rod,  and  in  this  way  give  to  the  rods  the  beaded 
appearance  so  commonly  ascribed  to  them. 

STAINING-PECULIARITIES. — A  peculiarity  of  this 
organism  is  its  behavior  toward  staining-reagents,  and 
by  this  means  alone  it  may  be  easily  recognized.  The 
tubercle  bacillus  does  not  stain  by  the  ordinary  methods. 
It  possesses  some  peculiarity  in  its  composition  that 
renders  it  proof  against  the  simpler  staining  processes. 
It  is  therefore  necessary  that  more  energetic  and  pene- 
trating reagents  than  the  ordinary  watery  solutions 
should  be  employed.  Experience  has  taught  us  that 
certain  substances  not  only  increase  the  solubility  of  the 
aniline  dyes,  but  by  their  presence  the  penetration  of 
the  coloring-agents  is  very  much  increased.  Two  of 
these  are  aniline  oil  and  carbolic  acid.  They  are 
employed  in  the  solutions  to  about  the  point  of  satura- 
tion. (For  the  exact  proportions,  see  chapter  on  Stain- 
ing-reagents.) 

Under  the  influence  of  heat  these  solutions  are  seen 
to  stain  all  bacteria  very  intensely — the  tubercle  bacilli 
as  well  as  other  forms.  If  we  subject  our  prepara- 
tion, which  may  contain  a  mixture  of  tubercle  bacilli 
and  other  species,  to  the  action  of  decolorizing-agents, 
another  peculiarity  of  the  tubercle  bacillus  will  be 
observed.  While  all  other  organisms  in  the  prepara- 
tion give  up  their  color  and  become  invisible,  the 
tubercle  bacillus  retains  it  with  marked  tenacity.  It 
stains  with  great  difficulty  ;  but  once  stained  it  retains 


ORGANISMS  RESEMBLING  £.   TUBERCULOSIS.   363 

the  color  even  under  the  action  of  strong  decolorizing- 
agents. 

We  have  reviewed  the  three  common  pathogenic 
organisms  that  may  be  encountered  in  the  sputum  of 
tuberculous  individuals.  Occasionally  other  species  may 
be  present.  The  pyogenic  forms  are  not  rarely  found, 
and  for  some  time  after  an  attack  of  diphtheria  the 
bacillus  of  Loffler  is  demonstrable  in  the  pharynx,  so 
that  it,  too,  may  be  present  under  exceptional  circum- 
stances. 

ORGANISMS    WITH    WHICH     BACTERIUM     TUBERCULOSIS 
MAY    BE    CONFUSED. 

It  is  important  to  note  that  in  the  study  of  tubercu- 
losis one  may  fall  into  error  unless  it  be  borne  in  mind 
that  there  is  a  group  of  bacilli  whose  members  are  in 
many  respects  so  like  the  genuine  bacillus  tuberculosis 
as  easily  to  be  mistaken  for  it.  While  its  peculiar 
micro-chemical  reaction  is  usually  sufficient  for  identifi- 
cation, particularly  in  connection  with  human  patho- 
logical lesions,  it  is  well  to  remember  that  the  confusing 
organisms  are  not  only  characterized  by  the  same  stain- 
ing peculiarities  as  bacillus  tuberculosis,  but  may  readily 
be  mistaken  for  it  on  morphological  grounds  also. 
Furthermore,  while  not  all  the  members  of  this  group 
are  capable  of  causing  disease,  some  of  them  are  patho- 
genic for  the  same  animals  that  are  susceptible  to  true 
tubercular  infection  ;  and  these  may  produce  in  those 
animals  lesions  which  are  distinguishable  from  genuine 
tubercles  only  by  their  finer  histological  structure.  A 
few  words  concerning  some  of  these  varieties,  with  a 
brief  summary  of  their  more  important  peculiarities, 
may  not  be  out  of  place. 


364  £.4  CTER10LOGY. 

BACTERIUM  LEPR^E. — Between  1879  and  1881  there 
was  described  by  Hansen  and  by  Neisser  an  organism, 
a  bacillus,  that  was  constantly  to  be  found  in  the  nodules, 
characteristic  of  leprosy.  For  this  organism  the  name  ba- 
cillus leprce  was  suggested.  Though  very  like  bacterium 
tuberculosis  in  both  morphology  and  staining  properties, 
it  is,  however,  a  little  shorter,  thicker,  and  muc!*  less 
homogeneously  stained.  Its  presence  in  the  tissues  and 
secretions  is  demonstrated  by  the  same  method  as  that 
employed  for  detecting  bacillus  tuberculosis.  In  sec- 
tions of  leprous  nodules,  stained  by  the  ordinary  Koch- 
Khrlich  process,  the  bacilli,  crowded  together  in  the 
large  so-called  "  lepra  cells/7  are  always  to  be  seen  in 
great  abundance.  It  is  unlikely  that  bacillus  leprse  has 
ever  been  cultivated  artificially,  and  the  disease  has  cer- 
tainly never  been  reproduced  in  animals  by  inoculation 
with  bits  of  the  diseased  tissue,  so  that  nothing  can  be 
said  of  the  life-history  of  this  organism. 

BACTERIUM  SMEGMATIS. — In  1885  Alvarez  and 
Tavel  discovered  in  the  fatty  secretions  about  the  gen- 
italia  an  organism  that  suggested  the  bacterium  of 
tuberculosis.  It  was  found  both  in  syphilitic  and  in 
healthy  persons.  Their  observation  has  been  abundantly 
confirmed  by  others,  and  the  organism  to  which  they 
directed  attention  is  now  regarded  as  pretty  commonly 
present  in  the  smegma.  It  is  known,  therefore,  as  the 
smegma  bacterium  (bacterium  smegmatis).  In  this 
secretion  it  is  found  in  clumps  located  upon  or  within 
epithelial  cells.  It  stains  by  the  method  used  in  staining 
bacterium  tuberculosis.  It  has  no  pathogenic  power. 
It  is  said  to  have  been  artificially  cultivated  upon  coag- 
ulated hydrocele  fluid  and  in  milk. 

In  1884  Lustgarten  described  an  organism,  the  so- 


THE  ACID-PROOF  BACTERIA.  365 

called  "bacillus  of  syphilis/7  that  he  had  discovered  in 
primary  syphilitic  lesions  and  in  the  secretions  from 
syphilitic  ulcers.  In  staining  reactions,  but  more  espe- 
cially in  morphology,  this  organism  is  said  to  be  stri- 
kingly like  bacterium  tuberculosis.  He  found  it  in  the 
tissues,  usually  within  the  bodies  of  large,  apparently 
swollen,  cells.  He  found  it  not  only  in  the  primary  sores 
about  the  genitalia,  but  in  the  syphilitic  lesions  of  the 
remote  organs  as  well.  As  this  organism  has  never 
been  cultivated  artificially,  and  as  the  majority  of  com- 
petent observers,  working  upon  the  most  promising 
material,  have  failed  to  detect  it,  the  prevailing  opinion 
is  to  the  effect  that  the  organism  is  not  regularly  asso- 
ciated with  syphilis  and  has  nothing  to  do  with  its 
causation. 

It  is  not  unlikely  that  bacterium  smegmatis  and  bac- 
terium syphilidis  are  identical. 

THE  ACID-PROOF  BACTERIA. — In  addition  to  the 
species  mentioned,  quite  a  group  of  other  "  acid-proof" 
bacteria,  as  they  are  called,  have  been  described  by  dif- 
ferent investigators.  They  are  characterized  by  staining, 
as  does  bacterium  tuberculosis,  by  retaining  the  stain 
to  a  greater  or  less  extent  when  treated  with  acids  and 
alcohol,  and  by  being  in  many  instances  strikingly  like 
bacterium  tuberculosis  in  their  morphology.  The  mem- 
bers of  this  group  seem  to  be  distributed  pretty  widely 
in  nature.  They  have  been  detected  in  non-tuberculous 
sputum,  in  gangrene  of  the  lung,  in  the  normal  intestinal 
contents  of  man  and  domestic  animals,  in  the  soil,  in 
fodder — i.  e.,  grass,  hay,  and  seed — in  manure,  and  in 
butter.  They  are  not  regularly  found  under  any  of  these 
conditions,  and  they  are  rarely  present  in  very  large 


366  BACTERIOLOGY. 

numbers.  Inasmuch  as  they  are  occasionally  encoun- 
tered under  circumstances  that  might  lead  one  to  look  for 
true  tubercle  bacilli,  and  since  they  possess  certain  pecu- 
liarities through  which  it  has  been  the  custom  to  identify 
bacillus  tuberculosis — /.  e.,  retention  of  the  stain  when 
acted  upon  by  acids  or  alcohol,  and  a  more  or  less  deli- 
cate, beaded  form — the  possibility  of  their  being  con- 
founded with  that  organism  is  obvious.  In  consequence 
they  have  received  a  great  deal  of  attention  during  the 
past  few  years. 

Space  does  not  permit  of  a  description  of  the  twenty 
odd  species  (?)  that  have  been  described  by  different  in- 
vestigators. It  will  suffice  to  say,  from  personal  study 
of  the  group,  that  in  all  probability  not  more  than  three? 
perhaps  only  two,  species  are  really  represented,  and 
that  the  remainder  may  fairly  be  regarded  as  varieties. 
As  said,  the  characteristic  common  to  all  the  mem- 
bers of  this  group  is  that  they  are  to  greater  or  less 
extent  acid-proof — i.  e.,  when  once  stained  by  the  Koch- 
Ehrlich  or  Ziehl  process  the  color  is  not  in  all  cases 
removed  by  the  ordinary  acid  decolorizers.  In  this  par- 
ticular, however,  there  is  considerable  variation.  In 
morphology  some  of  them  might  readily  be  mistaken 
for  bacillus  tuberculosis,  though  even  these  are  usually 
a  trifle  larger  and  less  delicate  than  that  organism ; 
others  are  at  once  differentiated  from  normal  tubercle 
bacilli,  but  have  somewhat  the  appearance  of  bacillus 
tuberculosis  when  degenerated  or  involuted ;  still  others 
have  nothing  in  their  general  appearance  to  lead  to  con- 
fusion. 

When  mixed  with  other  bacteria,  as  is  the  case  in  the 
soil,  in  manure,  in  intestinal  contents,  etc.,  their  isola- 
tion in  pure  culture  is  a  matter  of  difficulty,  and  this  is 


THE  ACID-PROOF  BACTERIA.  367 

by  no  means  lessened  by  the  fact  that  under  these  cir- 
cumstances they  are  always  numerically  in  the  minority. 
When  present  in  butter,  their  isolation  offers  fewer  diffi- 
culties, for  by  the  injection  of  the  butter  containing 
them  into  the  peritoneal  cavity  of  guinea-pigs  conditions 
are  created  that  favor  their  development,  and  from  ani- 
mals so  treated  they  may  usually  be  recovered  in  pure 
culture. 

When  studied  in  pure  culture,  all  of  them  are  at  once 
distinguished  from  bacillus  tuberculosis  by  the  follow- 
ing group  characteristics :  they  are  of  relatively  rapid 
growth,  there  being  usually  an  abundant  development 
on  glycerin  agar-agar  after  twenty-four  to  forty-eight 
hours  at  body-temperature ;  they  grow  well  but  less 
rapidly  at  ordinary  room-temperature — /.  <?.,  at  18°  to 
20°  C. ;  they  grow  well  in  litmus-milk,  and,  as  a  rule, 
produce  alkali  that  causes  the  color  to  become  a  deep 
blue ;  the  growth  on  agar-agar  is  dry,  shrivelled,  and 
wrinkled  in  appearance,  and  of  a  soft,  mealy  consistency 
in  some  cases  (Holler's  grass  bacillus  II.,  Rabinowitsch 
butter  bacillus,  for  instance),  while  in  others  it  is  more 
membranous,  as  in  the  case  of  Holler's  timothy  bacillus. 
We  have  never  seen  in  these  cultures  the  hard,  coarse 
granules  so  common  to  cultures  of  bacillus  tuberculosis  ; 
on  glycerin  agar-agar  some  of  them,  namely,  the  timothy 
bacillus  of  Holler  and  its  varieties,  grow  with  a  distinct 
orange  color,  while  others,  the  grass  bacillus  II.  of 
Holler,  the  butter  bacillus  of  Rabinowitsch,  and  their 
closely  allied  varieties,  begin  as  a  grayish-white  deposit 
which  may  ultimately  become  of  a  pale  or  even  distinct 
salmon  color. 

When  pure  cultures  of  them  are  injected  into  such 
animals  as  rabbits  or  guinea-pigs,  some  of  them  have  no 


368  BACTERIOLOGY. 

effect,  and  others  cause  lesions  of  more  or  less  impor- 
tance, the  result  being  dependent  upon  the  quantity  em- 
ployed and  the  mode  of  inoculation.  By  subcutaneous 
or  intraperitoneal  injection  of  pure  cultures  the  result  is 
usually  negative.  Occasionally  the  superficial  lymphatic 
glands  in  the  neighborhood  of  the  site  of  inoculation 
may  be  inflamed  and  purulent.  This  we  have  seen  only 
with  the  subcutaneous  inoculation.  If  pure  cultures  be 
injected  into  the  peritoneal  cavity  along  with  some 
sterile,  irritating  substance,  such  as  sterilized  butter,  a 
widespread  fibrinopurulent  peritonitis  is  commonly  the 
result. 

When  injected  directly  into  the  circulation  of  rabbits, 
the  kidneys  are  almost  uniformly  affected,  and  in  the 
majority  of  instances  they  are,  singularly  enough,  the 
only  organs  in  which  lesions  are  to  be  detected.  If,  for 
instance,  a  cubic  centimetre  of  a  carefully  prepared 
suspension  in  bouillon  of,  let  us  say,  Moller's  grass 
bacillus  II.,  be  injected  into  the  circulation  of  a  rabbit, 
and  the  animal  be  killed  after  twelve  to  fourteen  days, 
the  kidneys  will  be  found  marked  by  gray  or  yellowish 
points  that  range  in  size  from  that  of  a  pin-point  to 
that  of  a  pin-head.  They  are  sometimes  very  few  in 
number,  but  in  other  cases  both  kidneys  may  be  thickly 
studded  with  them.  Often  they  are  not  elevated  above 
the  cortex  of  the  organ,  but  in  as  many  cases  they  are 
sharply  defined,  yellow  in  color,  and  stand  up  promi- 
nently from  the  cortical  surface,  being  at  the  same  time 
so  adherent  to  the  capsule  that  the  removal  of  the  latter 
tears  them  out  bodily  from  the  substance  of  the  organ. 
In  the  very  early  stages  of  development  these  nodules 
are  often  difficult  to  distinguish  from  young  tubercles,  the 
reaction  of  the  tissues  being,  as  in  the  case  of  tubercles, 


THE  ACID-PROOF  BACTERIA.  369 

characterized  by  proliferation  of  the  fixed  cells  with 
little  evidence  of  leucocytic  invasion ;  later  on,  true 
giant-cell  formation  is  recorded  by  some  observers.  We 
have  not  seen  this.  Clumps  of  endothelial  nuclei  or  of 
lymphoid  cells  that  remotely  suggest  the  arrangement 
seen  in  giant  cells  are  often  encountered,  but  we  have 
not  regarded  them  as  true  giant  cells.  When  fully 
developed,  the  nodule  may  present  a  mixed  condition  of 
caseation  and  suppuration.  The  conditions,  as  a  whole, 
when  advanced  suggest  a  low  grade  of  inflammatory 
reaction.  Occasionally  nodules  are  encountered,  espe- 
cially in  the  kidney,  that  cannot  be  distinguished  from 
tubercles.  The  bacilli  are  always  to  be  found  within 
the  nodules ;  most  frequently  as  single  rods  or  clumps 
of  rods,  occasionally  as  rosette-like  mycelia  very  sug- 
gestive of  the  characteristic  growth  of  the  actinomyces 
fungus  in  the  tissues.  This  mode  of  development  has 
also  been  observed  with  bacillus  tuberculosis. 

It  is  important  to  note  the  difference  between  the  re- 
sults of  intravenous  inoculation  of  rabbits  with  bacillus 
tuberculosis  and  with  the  organisms  under  consideration. 
When  bacillus  tuberculosis  is  employed,  the  lungs,  as 
well  as  the  kidneys,  are  always  involved,  while  with  the 
grass  bacillus  II.,  the  timothy  bacillus,  and  the  butter 
bacillus,  involvement  of  the  lungs,  in  our  experiments, 
has  been  the  exception  rather  than  the  rule. 

Another  point  of  interest  is  the  lack  of  tendency  on 
the  part  of  the  non-tuberculous  process  to  progress  or 
become  disseminated. 

That  the  members  of  this  group  are  botanically 
related  to  bacillus  tuberculosis  there  seems  little  room 
to  doubt ;  but  from  personal  study  and  from  available 
evidence  from  other  sources  it  appears  unlikely  that 

24 


370  BACTERIOLOGY. 

they  are,  except  experimentally,  concerned  in  disease- 
production  or  that  they  are  of  importance  to  either 
human  or  animal  pathology.1 

In  the  microscopic  examination,  particularly  of  urine, 
of  secretions  from  about  the  anus,  rectum,  and  genitalia, 
and  of  butter,  it  is  manifestly  of  importance  to  bear  in 
mind  the  existence  of  this  confusing  group,  for  it  is  in 
such  secretions  and  substances  that  they  are  most  often 
encountered.  The  smegma  bacillus  and  the  butter 
bacillus  are  especially  liable  to  lead  one  into  error  of 
diagnosis.  This  is  less  apt  to  be  the  case  with  the  com- 
paratively rare  lepra  bacillus  and  the  questionable 
syphilis  bacillus. 

DIFFERENTIAL  DIAGNOSIS. — According  to  Hueppe, 
the  differential  diagnosis  between  bact.  tuberculosis, 
bact.  smegmatis,  and  bact.  leprse,  depends  upon  the  fol- 
lowing reactions:  when  stained  by  the  carbol-fuchsin 
method  commonly  employed  in  staining  the  tubercle 
bacillus  the  syphilis  bacillus  becomes  almost  instantly 
decolorized  by  treatment  with  mineral  acids,  particularly 
sulphuric  acid,  whereas  the  smegma  bacillus  resists  such 
treatment  for  a  much  longer  time,  and  the  lepra  and 
tubercle  bacillus  for  a  still  longer  time.  On  the  other 
hand,  if  decolorization  is  practised  with  alcohol,  instead 
of  acids,  the  smegma  bacillus  is  the  first  to  lose  its  color. 
The  bacillus  tuberculosis  and  the  bacillus  of  leprosy  are 
conspicuously  retentive  of  their  color  even  after  treat- 
ment with  both  acids  and  alcohol.  To  differentiate, 
then,  between  the  four  organisms  he  recommends  the 
following  order  of  procedure,  based  on  the  above  reac- 
tions : 

xFor  the  literature  on  "acid-proof"  bacilli,  see  Cowie,  Journal  of 
Experimental  Medicine,  1900,  vol.  v.  p.  205. 


DIFFERENTIAL  DIAGNOSIS.  371 

1.  Treat  the  preparation,  stained  with  carbol-fuchsin, 
with  dilute  sulphuric  acid ;  the  so-called  syphilis  bacillus  be- 
comes decolorized,  the  reaction  being  almost  instantaneous. 

2.  If  it  is  not  at  once  decolorized,  treat  with  alcohol  ; 
if  it  is  the  smegma  bacillus,  this  will  rob  it  of  its  color. 

3.  If  it  is  still  not  decolorized,  it  is  either  the  lepra 
or  tubercle  bacillus. 

Grethe  recommends l  the  following  as  a  trustworthy 
means  of  distinguishing  between  the  tubercle  bacillus  and 
the  smegma  bacillus  :  stain  in  hot  carbol-fuchsin  solution, 
wash  in  water,  and  treat  the  preparation  with  a  saturated 
solution  of  methylene-blue  in  alcohol.  If  the  question- 
able organism  is  the  tubercle  bacillus,  it  retains  its  red 
color ;  if  the  smegma  bacillus,  the  red  color  is  dissolved 
by  the  alcohol  and  the  blue  color  is  substituted  for  it. 

The  differential  diagnosis  between  the  tubercle  bacil- 
lus and  the  lepra  bacillus  is  less  satisfactory ;  they  both 
take  the  same  stains,  and  both  retain  them  or  give 
them  up  under  treatment  with  the  same  decolorizers. 
The  results  of  investigations,  however,  indicate  differ- 
ences in  the  rate  of  staining  and  decolorization,  and  it  is 
stated  by  many  of  those  who  have  compared  the  two 
organisms  that  the  lepra  bacillus  takes  up  stain  very 
much  more  readily  than  does  the  tubercle  bacillus, 
often  staining  perfectly  after  an  exposure  of  only  a  few 
minutes  to  cold  watery  solutions  of  the  dyes ;  but  when 
once  stained  it  retains  its  color  much  more  tenaciously 
when  acted  upon  by  decolorizing-agents  than  does  the 
latter  organism. 

According  to  Baumgarten,  the  lepra  bacillus  is  stained 
by  an  exposure  of  six  to  seven  minutes  to  a  cold,  satu- 
rated watery  solution  of  fuchsin,  and  retains  the  stain 

1  Fortschritte  der  Medicin,  1896,  No.  9. 


372  BACTERIOLOGY. 

when  subsequently  treated  with  acid  alcohol  (nitric 
acid,  1  part ;  alcohol,  10  parts).  By  similar  treatment 
for  the  same  length  of  time  bacillus  tuberculosis  does 
not  ordinarily  become  stained. 

These  points,  particularly  what  has  been  said  with  ref- 
erence to  the  smegma  bacillus,  are  of  much  practical 
importance,  and  should  always  be  borne  in  mind  in  con- 
nection with  the  microscopic  examination  of  materials 
to  which  these  organisms  are  likely  to  gain  access.  It 
is  hardly  necessary  to  say  that  in  the  examination  of 
sputum  and  pathological  fluids  from  other  parts  of  the 
body  the  tubercle  bacillus  is,  of  the  organisms  noted, 
always  the  one  most  commonly  encountered,  while  the 
organism  described  by  Lustgarten  as  the  bacillus  of 
syphilis  is  seen  so  rarely  that  many  trustworthy  investi- 
gators question  its  existence  as  a  species  distinct  from 
the  ordinary  smegma  bacillus. 

BACTERIUM    TUBERCULOSIS    AVIUM  (MAFFUCCl), 
MIGULA,  1900. 

Synonyms:  Bacillus  tuberculosis  avium,  Maffucci,  1891 ;  Mycolmcte- 
rium  tuberculosis  avium,  Lebrnann  and  Neumann,  1896. 

From  time  to  time  fowls  are  known  to  suffer  from 
a  form  of  tuberculosis  that  in  a  number  of  ways  sug- 
gests human  or  mammalian  tuberculosis.  The  bacillus 
causing  the  disease,  the  so-called  bacillus  of  fowl  tuber- 
culosis, bacillus  tuberculosis  avium,  while  simulating  the 
genuine  bacillus  tuberculosis  morphologically,  differs 
from  it  both  in  cultural  and  pathogenic  peculiarities. 
Thus,  for  instance,  it  develops  into  much  longer  and 
somewhat  thinner  threads ;  grows  rapidly  on  media 
without  glycerin  or  glucose ;  does  not  grow  on  potato  ; 
develops  as  well  at  from  42°  to  43°  C.  as  at  37°  to  38° 


BACTERIUM  TUBERCULOSIS  AV1UM.          373 

C.;1  its  virulence  is  not  diminished  by  cultivation  at 
43°  C. ;  development  on  artificial  media  begins  in  from 
six  to  eight  days  after  inoculation  ;  young  cultures  on 
solid  media  are  whitish,  soft,  and  moist,  becoming  yel- 
lowish and  slimy  with  age ;  it  is  somewhat  more  resist- 
ant to  drying  and  high  temperatures  than  the  bacillus 
of  mammalian  tuberculosis;  the  results  of  its  patho- 
genic activities  are  almost  always  chronic,  are  rarely 
located  in  the  lungs  or  intestines,  but  are  especially  fre- 
quent in  the  liver  and  spleen  ;  the  lesions  are  conspic- 
uously rich  in  bacteria,  do  not  show  the  central  necrotic 
area  that  characterizes  the  mammalian  tubercle ;  the 
disease  is  transmissible  from  the  hen  to  the  embryo 
chick;  the  only  susceptible  mammal  is  the  rabbit;  the 
guinea-pig  and  dog  are  naturally  immune ;  it  has  the 
same  micro-chemical  staining-reactions  as  mammalian 
bacillus  tuberculosis;  it  has  never  been  certainly  de- 
tected in  human  tuberculosis. 

Some  are  inclined  to  regard  this  organism  as  but  a 
variety  of  the  genuine  bacillus  tuberculosis,  and  it  is  not 
unreasonable  to  believe  that  the  sojourn  of  that  organ- 
ism in  the  body  of  a  refractory  animal,  whose  normal 
temperature  is  so  high  as  that  of  the  fowl,  when  not  fatal 
to  the  organism,  might  result  in  striking  modifications 
of  certain  of  its  biological  functions.  In  fact,  Nocard 2 
has  shown  that  if  the  genuine  bacillus  tuberculosis  from 
man  be  left  in  the  peritoneal  cavity  of  chickens  (by  the 
collodion-sac  method  of  Metschnikoff,  Roux,  and  Sal- 
lembini,  which  see)  for  from  five  to  eighth  months,  they 
will,  by  the  end  of  this  time,  have  become  so  modified 

1  The  normal  body-temperature  of  fowls  ranges  between  41.5°  and 
42.">°  C. 

-  Nocard:  Anntiles  de  1'Institut  Pasteur,  1898,  p.  f>61. 


374  BA  CTERIOLOG  Y. 

in  their  biological  peculiarities  as  to  simulate  very 
closely  the  bacillus  of  fowl  tuberculosis. 

Moore1  reports  studies  on  bacterium  tuberculosis 
avium  iu  an  epidemic  occurring  in  California.  He  ob- 
tained pure  cultures  by  inoculating  glycerin-agar  or  blood- 
serum  tubes  directly  from  tuberculous  livers  and  spleens. 
In  the  original  cultures  little  difficulty  was  experienced 
in  cultivating  the  organism  on  glyeerine-agar,  fresh  dog- 
serum,  Dorset's  egg-medium,  potato,  and  glycerine- 
bouillon.  The  general  cultural  peculiarities  observed 
agreed  with  those  described  by  Maffucci,  Nocard,  Straus 
and  Gamaleia,  and  others.  He  states  that  the  tubercle 
bacteria  resemble  quite  closely  those  of  the  bovine  and 
human  varieties  in  their  size  and  general  morphology 
as  they  are  found  in  the  tissues  of  the  fowl.  The  aver- 
age length  of  a  large  number  of  measurements  was  2.7 
microns.  Moore  also  tested  the  pathogenesis  of  the 
freshly  isolated  avian  tubercle  bacteria  on  fowls,  rabbits, 
guinea-pigs,  and  pigeons.  The  results  of  these  inocu- 
lations, however,  were  unsatisfactory,  as  were  also  feed- 
ing experiments  of  healthy  fowls  with  human  tubercu- 
lous sputum  rich  in  bacteria. 

VARIETIES  OF  B.  TUBERCULOSIS. — Theobald  Smith2 
has  called  attention  to  certain  very  conspicuous  differences 
that  maybe  observed  between  the  bacilli  obtained  from  hu- 
man and  those  from  bovine  tuberculosis ;  and  in  a  series  of 
inoculation  experiments  Ravenel  has  shown  that  for  a  large 
number  of  species  tubercle  bacilli  of  bovine  origin  were 
constantly  more  virulent  than  those  from  human  sources. 

Anatomical   lesions   very  suggestive  of,  though  not 

1  Moore:  Journal  of  Medical  Eesearch,  1904,  vol.  vi. 

2  Smith :  Transactions  of  the  Association  of  American  Physicians, 
1896,  vol.  xi.  p.  75. 


TUBERCULIN.  375 

identical  with,  those  produced  by  bacillus  tuberculosis, 
have  also  from  time  to  time  been  observed  in  mice,  rats, 
guinea-pigs,  rabbits,  cats,  goats,  bovines,  hogs,  and  man. 
They  do  not  appear  to  be  of  a  specific  nature  as  regards 
etiology,  for  the  reason  that  different  authors  have 
described  different  organisms  as  the  causative  agents. 
These  affections  are  usually  classed  under  the  name 
pseudotuberculosis. 

SUSCEPTIBILITY  OF  ANIMALS  TO  TUBERCULOSIS. — 
The  animals  that  are  known  to  be  susceptible  to  tuber- 
culosis are  man,  apes,  cattle,  horses,  sheep,  guinea-pigs, 
pigeons,  rabbits,  cats,  and  field-mice.  White  mice, 
dogs,  and  rats  possess  immunity  from  the  disease. 

TUBERCULIN. — The  filtered  products  of  growth  from 
old  fluid  cultures  of  the  tubercle  bacillus  represent  what 
is  known  as  tuberculin — a  group  of  proteid  substances 
possessing  most  interesting  properties.  When  injected 
subcutaneously  into  healthy  subjects  tuberculin  has  no 
effect ;  but  when  introduced  into  the  body  of  a  tuber- 
culous person  or  animal  a  pronounced  systemic  reaction 
results,  consisting  of  sudden  but  temporary  elevation  of 
temperature,  with,  at  the  same  time,  the  occurrence 
of  marked  hypersemia  about  the  tuberculous  focus, 
a  change  histologically  analogous  to  that  seen  in  the 
primary  stages  of  acute  inflammation.  This  zone  of 
hypersemia,  with  the  coincident  exudation  and  infiltra- 
tion of  cellular  elements,  probably  aids  in  the  isolation 
or  casting  off  of  the  tuberculous  nodule,  the  inflamma- 
tory zone  forming,  so  to  speak,  a  line  of  demarcation 
between  the  diseased  and  healthy  tissue. 

As  a  curative  agent  for  the  treatment  of  tuberculosis, 
tuberculin  has  not  merited  the  confidence  that  was  at 


376  BACTERTOLOG  Y. 

first  accorded  to  it.  Its  field  of  usefulness  is  now 
almost  limited  to  the  diagnosis  of  obscure  cases,  and 
even  for  this  purpose  it  is  less  frequently  employed 
than  formerly. 

In  veterinary  medicine  it  has  proved  much  more 
trustworthy  as  a  diagnostic  aid,  and  is  practically 
everywhere  in  use  for  the  detection  of  incipient  tuber- 
culosis, especially  in  cattle. 

VACCINATION  AGAINST  TUBERCULOSIS. — Recent 
experiments  by  v.  Behriiig,  Pearson  and  Gilliland,  and 
others  have  shown  that  it  is  possible  to  immunize  ani- 
mals with  lowly  virulent  tubercle  bacteria  of  human 
origin,  and  after  one  or  two  injections  with  such  organ- 
isms the  animals  show  a  marked  degree  of  tolerance  to 
the  more  highly  virulent  bovine  organisms.  The 
results  of  experiments  in  this  direction  have  been  so 
encouraging  that  it  is  probable  this  method  may  be 
utilized  for  the  active  immunization  of  cattle  against 
tuberculosis. 

ACTINOMYCETES. 

The  term  actinomycetes  is  restricted  to  a  group  of 
organisms  having  morphological  affinities  with  the  bac- 
teria on  the  one  hand  and  the  hyphomycetes  on  the 
other.  They  resemble  the  bacteria  in  that  they  occur 
as  homogeneous  threads  which  under  artificial  cultiva- 
tion may  become  segmented  into  short  bacillus-  or 
coccus-like  fragments.  Furthermore,  they  are  unlike 
the  moulds  in  that  they  have  not  a  double  wall ;  are  not 
filled  with  fluid  containing  granules,  and  the  segments 
are  not  separated  from  one  another  by  a  distinct  parti- 
tion. They  simulate  the  moulds  in  that  they  develop 
from  spores  into  dichotomously  branching  threads, 


A  CTINOMYCETES.  37  7 

which  ultimately  form  colonies  having  more  or  less 
resemblance  to  true  mycelia.  Certain  of  the  threads 
composing  such  a  mycelium  become  fruit  hyphse,  break- 
ing up  into  round,  glistening,  spore-like  bodies.  As  a 
rule,  these  spores  are  devoid  of  the  high  resistance  to 
heat  exhibited  by  bacterial  spores,  and  are  stainable  by 
the  ordinary  methods. 

The  limits  of  this  group  are  ill  defined  and  its  recog- 
nized components  are  not  as  a  whole  well  understood. 

The  longest  known  and  most  carefully  studied  acti- 
nomycetes  are  act.  bovis,  act.  madurse,  act.  farcinicus, 
ard  act.  Eppingeri,  although  many  other  varieties  have 
been  encountered  in  association  with  important  and 
interesting  pathological  lesions. 

The  fact  that  certain  bacteria,  viz.,  b.  tuberculosis,  b. 
mallei,  b.  diphtheria,  generally  regarded  as  bacteria, 
are,  as  a  rule,  segmented  and  occasionally  show  a  ten- 
dency to  branch,  has  led  to  their  being  classified  at 
times  with  the  actinomycetes ;  on  this  point,  however, 
there  is  as  yet  no  consensus  of  opinion. 

It  is  interesting  to  note  that  the  pathological  lesions 
in  which  actinomycetes  have  been  detected  show  in  many 
cases  certain  similarities  to  true  tubercular  processes, 
and  in  a  few  instances,  save  for  the  absence  of  tubercle 
bacteria,  as  we  usually  see  them,  were  indistinguishable 
from  tuberculosis. 

More  or  less  imperfectly  studied  varieties  of  actino- 
mycetes have  been  encountered  in  abscess  of  the  brain, 
cerebrospinal  meningitis,  endocarditis,  bronchopneumo- 
nia,  pleuropneumonia,  pustular  exanthemata,  abscess  of 
the  lung,  bronchiectasis,  pulmonary  gangrene,  necrosis 
of  the  vertebra,  subphrenic  abscess,  noma,  pseudotuber- 
c,ulosis,  etc. 


378  BACTERIOLOGY. 

In  some  cases  the  actinomycetes  can  be  obtained  in 
culture  from  the  diseased  tissues ;  almost  as  often  they 
cannot.  Sometimes  the  inoculation  of  animals  with 
bits  of  the  diseased  tissue  or  with  cultures  results  in  the 
production  of  pathological  lesions  referable  to  the  organ- 
ism ;  again,  no  effect  follows  upon  such  inoculation.  As 
seen  in  the  tissues  by  microscopic  examination,  actino- 
mycetes may  appear  as  long,  convoluted,  irregularly  stain- 
ing, beaded,  branching  threads,  or  as  clumps  of  short, 
markedly  beaded,  sometimes  branched  rods.  At  times 
a  clump  of  the  short  or  longer  threads  is  encountered 
in  the  tissues  that  gives  the  distinct  impression  of 
mycelial  structure. 

Some  of  the  varieties  that  have  been  described  are 
best  demonstrated  in  the  tissues  or  exudates  by  the 
Gram  or  Gram-Weigert  method  of  staining ;  others  are 
decolorized  by  this  process,  and  are  rendered  visible 
only  by  the  simpler  procedures.  Some  of  them  are  to 
a  limited  extent  proof  against  the  action  of  acid  decolor- 
izers.  Though  many  accounts  of  the  presence  of  these 
morphological  types  in  a  variety  of  conditions  have 
been  recorded,  the  descriptions  in  the  main  are  meagre 
and  often  insufficient  for  identification.  A  few,  how- 
ever, have  been  found  so  constantly  in  association  with 
more  or  less  definite  clinical  and  pathological  conditions 
that  a  brief  description  of  them  may  be  of  service. 

ACTINOMYCES  BO  vis  (also  commonly  known  as  strep- 
tothrix  actinomyces,  actinomyces  fungus,  ray  fungus) 
was  first  observed  by  von  Langenbeck  in  a  case  of 
vertebral  caries  in  1845.  According  to  Bellinger,  the 
fungus  had  been  seen  by  Hahn  a  number  of  years  before 
in  museum  specimens,  but  had  been  regarded  by  him 
as  a  penicillium.  The  name  actinomyces  or  ray  fungus 


A  CTINOMYCETES.  379 

originated  with  Harz.  It  is  constantly  to  be  detected  in 
the  tissues  and  exudates  of  the  disease  of  cattle  known 
as  actinomycosis,  "  lumpy  jaw,"  "  wooden  tongue,"  etc. 
The  typical  tumor  of  this  disease  is  characterized  by 
inflammation,  pus  formation,  excessive  new  formation 
of  connective  tissue,  abscesses,  cavities,  and  sinuses. 
Viewed  as  a  whole,  the  tumor  presents  points  of  resem- 
blance to  the  osteo-sarcomatous,  the  scrofulous  or  tuber- 
culous, and  the  cancerous  processes.  The  disease  occa- 
sionally occurs  in  man,  and  according  to  the  point  of 
entrance  of  the  parasite  may  arise  in  the  mouth,  the 
pharynx,  the  lungs,  the  intestines,  or  the  skin.  In  ani- 
mals the  disease  is  characterized  by  an  excessive  new 
formation  of  connective  tissue,  so  that  tumefaction  is 
always  a  conspicuous  peculiarity.  In  man,  on  the  other 
hand,  suppuration  is  the  most  prominent  feature. 

If  the  purulent  discharge  from  an  actinomycotic 
tumor  be  examined  fresh,  it  will  be  found  to  contain 
tiny  yellow  (sulphur  color  as  a  rule)  clumps.  If  these 
be  examined,  unstained,  in  a  drop  of  physiological  salt 
solution  or  water  under  the  microscope,  they  will  be  found 
to  be  made  up  of  a  rosette-like  mass  of  closely  inter- 
woven threads.  (See  Fig.  65.)  At  the  centre  the  mass 
may  show  the  presence  of  spherical,  coccus-like  bodies 
or  granules,  while  at  the  periphery  the  free  ends  of  the 
threads  are  more  or  less  distinctly  bulbous  or  nodular, 
or  both,  and  they  may  show  branching.  Sometimes  the 
free  ends  of  the  threads  are  only  slightly  or  not  at  all 
swollen. 

These  mycelia — the  actinomyces — may  be  stained  by 
the  ordinary  aniline  dyes,  or  by  the  Weigert  or  tho 
Gram  method,  though  by  either  of  these  procedures  its 
full  structure  is  not,  as  a  rule,  brought  out.  The  reason 


380  BA  CTERTOLOG  Y. 

for  this  is  that  the  terminal  bulbs  are  not  due  to  enlarge- 
ment of  the  thread  itself,  but  rather  to  a  colloid  degen- 
eration of  its  enveloping  sheath.  This  colloid  matter, 
having  different  microchemical  reactions  from  the 
enclosed  thread,  requires  different  reagents  to  stain  it. 
The  entire  structure  may  be  seen  when  the  fungus  is 
stained  first  by  the  Gram  method,  and  subsequently  with 
eosin  or  saffranin.  For  the  demonstration  of  the  fungus 
in  sections,  the  method  of  Mallory  gives  satisfaction. 
It  is  as  follows :  Stain  the  section  on  the  side  with 

FIG.  65. 


Actinomycosis  fungus  in  pus.    Fresh,  unstained  preparation.    Magnified 
about  500  diameters. 

gentian-violet ;  clear  and  dehydrate  with  aniline  oil  in 
which  a  little  basic  fuchsin  has  been  dissolved ;  remove 
the  aniline  oil-fuchsin  with  xylol,  and  mount  in  xylol 
balsam.  In  sections  treated  in  this  way  the  coccus-like 
central  masses  and  the  filamentous  threads  making  up 
the  mass  of  the  mycelium  are  stained  blue ;  the  club- 
like  extremities  of  the  thread  are  red.  Often  the  red- 
stained  hyaline  material  is  seen  to  be  penetrated  to  its 
extremity  by  a  sharply  defined  blue  thread. 

Cultivation  of  the  fungus  from  the  actinomycotic  pus 


A  CTINOM  YCETES.  3  8 1 

presents  difficulties  for  the  following  reasons  :  All  the 
mycelia  seen  by  microscopic  examination  are  not  living  ; 
as  a  rule,  they  grow  slowly  even  under  the  best  of  cir- 
cumstances ;  and  generally  there  are  many  other,  more 
rapidly  growing,  living  organisms  in  the  pus.  When 
pure  cultures  are  obtained,  it  grows  (according  to  Bos- 
trom)  on  all  the  ordinary  artificial  media.  It  develops 
at  room-temperature,  but  better  at  that  of  the  body. 

It  grows  both  with  and  without  oxygen. 

The  young  colonies  appear  as  grayish  points  com- 
posed of  a  felt- work  of  fine  threads.  As  the  colonies 
become  older  they  become  denser  and  more  opaque. 
Very  old  colonies  are  almost  leathery  in  consistency. 
On  blood-serum  the  growth  after  a  time  assumes  a 
salmon,  an  orange,  or  a  yellowish-red  color.  Growth 
on  gelatin  is  accompanied  by  slow  liquefaction. 

A  yellowish-red  growth,  limited  in  extent,  occurs  on 
potato.  It  causes  no  clouding  of  bouillon,  but  grows  as 
cottony  clumps  that  sink  to  the  bottom. 

The  bulbous  extremities  seen  upon  the  mycelial 
threads  fresh  from  the  pus  are  not  usually  seen  under 
conditions  of  artificial  cultivation.  They  are  sometimes 
observed  in  colonies  located  in  the  depths  of  solid  media. 
The  white,  powdery  coating  seen  on  old  colonies  repre- 
sents the  so-called  "  spores."  They  are  not,  however, 
resistant  to  heat,  being  destroyed,  according  to  Domec, 
by  75°  C.  in  five  minutes. 

Bovines  are  the  animals  most  frequently  affected. 
The  disease  has  been  seen  in  swine,  dogs,  and  horses. 

The  most  common  seat  of  the  disease  is  the  jaw,  and 
this,  together  with  the  fact  that  particles  of  fodder,  such 
as  bits  of  grain,  chaff,  straw,  and  barley  beard,  have 
been  detected  in  the  diseased  tissues  in  association  with 


382  BACTERIOLOGY. 

the  causative  fungus,  has  led  to  the  belief  that  the  par- 
asite gains  access  to  the  tissues  with  such  food-stuffs.  It 
has  not,  however,  been  recognized  outside  the  animal  body. 
The  disease  is  apparently  not  transmissible  from  animal 
to  animal  or  from  animal  to  man.  Inoculation  of  ani- 
mals with  pure  cultures  is  usually  negative,  although 
nodular  formations  have  followed  the  injection  of  large 
quantities  into  the  peritoneal  cavity  of  rabbits.  In 
Bostrom's  cases  the  nodules  presented  only  a  few  of  me 
club-shaped  extremities  of  the  threads,  and  there  was 
no  evidence  of  multiplication  of  the  fungus ;  while  in 
the  experiments  of  Israel  and  Wolf  it  is  said  there 
developed,  in  from  four  to  seven  weeks  after  intraperi- 
toneal  inoculation,  larger  and  smaller  tumors  in  which 
typical  mycelia  were  present,  and  from  which  the  fungus 
was  obtained  in  pure  culture. 

ACTINOMYCES  MADURA. — This  organism  is  sup- 
posed to  be  concerned  in  the  causation  of  mycetoma  or 
Madura  foot.  Two  varieties  of  mycetoma  are  known, 
viz.,  the  pale  or  ochroid  and  the  black  or  melanoid. 
Save  for  its  occurrence  in  the  foot,  mycetoma  is,  patho- 
logically speaking,  almost  a  counterpart  of  actinomy- 
cosis ;  and  the  suspicion  of  their  identity  is  by  no 
means  lessened  by  the  fact  that  the  actinomyces  con- 
stantly associated  with  the  ochroid  variety  is  to  all 
intents  and  purposes  identical  with  actinomyces  bovis. 
It  differs  from  that  organism  only  in  such  minor  details 
as  to  leave  little  doubt  that  they  are  very  closely  related, 
if  not  identical,  so  that  a  description  of  the  one  serves 
equally  to  aid  in  the  identification  of  the  other. 

The  investigations  of  Wright,1  conducted  upon  a  case 

1  Wright :  "  A  Case  of  Mycetoma  (Madura  Foot),"  Journal  of  Experi- 
mental Medicine,  1898,  vol.  in.,  p.  421. 


ACTINOMYCETES.  283 

encountered  in  Boston,  point  to  another  type  of  parasite  as 
the  causative  factor  in  the  black  mycetoma.  Instead  of  an 
actinomyces,  Wright  found  a  true  mould.  He  expresses 
the  opinion  that  the  pale  mycetoma  is,  etiologically,  actino- 
mycosis,  and  that  the  black  is  a  hyphomycetic  infection. 

The  fungus  encountered  and  isolated  in  pure  culture 
by  Wright  presented  the  following  characteristics  :  As 
obtained  from  the  affected  tissues,  the  mycelia  under  the 
microscope  appear  as  black  or  brown  mulberry-like 
masses  less  than  one  millimetre  in  diameter.  They  are 
hard,  rather  brittle,  and  difficult  to  break  up  under  the 
cover-glass.  On  soaking  them  in  a  strong  solution  of 
sodium  hydroxide  they  become  softened  and  the  struct- 
ure of  the  fungus-mass  can  be  made  out.  Under  high 
magnifying  power  these  masses  are  found  to  consist  of 
pigment-granules,  ovoid  translucent  bodies,  and  dis- 
tinctly branching  separate  hyphse.  Sometimes  these 
latter  exhibit  dilatations  or  varicosities  of  their  seg- 
ments. The  periphery  of  a  granule  shows  the  pres- 
ence of  club-shaped  hyphse,  closely  set  and  radially 
arranged.  From  such  granules  growth  on  artificial 
culture-media  may  be  obtained.  When  transferred 
direct  from  the  tissues  to  artificial  media,  growth  in 
every  case  starts  from  the  granule  about  four  or  five 
days  after  it  is  placed  upon  the  culture-media. 

On  solid  media  it  first  appears  as  delicate  tufts  of 
whitish  filaments.  These  in  the  course  of  a  few  days 
increase  in  number  and  length,  and,  in  the  case  of  the 
potato,  form  a  dense  whitish  or  pale-brown  felt-work 
having  a  tendency  to  spread  widely. 

In  pure  cultivation  it  is  seen  as  long,  branching  hyphse 
with  delicate  transverse  septa.  In  old  forms  the  hyphse 
may  be  swollen  at  the  points  marked  by  the  septa,  and 


384  BACTERIOLOGY. 

may  then  appear  as  strings  of  plump  oval  segments. 
The  filaments  have  a  definite  wall,  inclosing  granules 
and  pale  areas.  No  spore-bearing  organs  are  seen. 

On  potato,  it  grows  as  a  dense,  widely  spreading,  vel- 
vety membrane;  pale  brown  at  the  centre  and  white  at 
the  periphery.  The  potato  takes  on  a  dark-brown  color 
and  becomes  very  moist  and  dark ;  coffee-colored  gran- 
ules appear  upon  the  surface  of  the  growth. 

In  bouillon  the  growth  assumes  a  puff-ball  appear- 
ance. The  medium  assumes  a  deep  coffee-brown  color, 
and  ultimately  a  mycelium  growth  appears  upon  the 
surface  and  throughout  the  fluid. 

When  grown  in  potato  infusion  (20  grammes  of  potato 
boiled  in  water,  filtered  and  made  up  to  a  litre),  the 
growth  is  characterized  by  the  appearance  of  black 
granules  in  the  midst  of  the  mycelium.  The  black 
granules  consist  of  closely  packed  spherical  or  polyhe- 
dral cells,  together  with  some  short,  thick  segmented 
hyphse.  The  walls  of  these  cells  have  a  black  appear- 
ance, and  masses  of  them  are  black  and  opaque  under 
the  microscope. 

On  agar-agar,  growth  appears  as  a  grayish  mesh-work 
of  widely  spreading  filaments.  In  old  cultures  black 
granules  (sclerotia)  appear  among  the  filaments.  No 
growth  occurs  in  the  depth  of  the  medium. 

No  results  were  obtained  by  the  inoculation  of  ani- 
mals with  either  the  material  direct  from  the  tissues  or 
with  pure  cultures. 

The  tissue  from  which  this  fungus  was  obtained  con- 
sisted, briefly,  of  a  more  or  less  atypical  connective- 
tissue  new-growth,  with  numerous  areas  of  suppuration 
marked  by  the  black  granules  just  described. 

On  histological  study  of  the  tumor  the  primary  effect 


ACTINOMYCETES.  385 

produced  by  the  parasite  appears  to  be  the  development 
of  nodules  of  epithelial  cells  and  of  giant  cells  from  the 
tissues  immediately  about  them.  Later,  suppuration  of 
the  nodules  and  abscess  formation  occur.  This  in  time 
gives  rise  to  excessive  development  of  granulation  and 
connective  tissue. 

ACTINOMYCES  FARCiNicus  (bacille  du  farcin  des 
boeuffs  (Nocard)  ;  oospora  farcinica ;  actinomyces  bovis 
farcinicus). — This  organism  was  discovered  by  Nocard 
(1888)  in  a  disease  of  cattle  that  is  suggestive  of  farcy 
as  seen  in  horses.  The  lesions  consist  of  chains  of  en- 
larged subcutaneous  lymph-glands,  which  on  examina- 
tion are  found  to  be  in  a  condition  somewhat  simulating 
tuberculosis.  Similar  nodules  are  sometimes  encoun- 
tered in  the  internal  organs. 

By  microscopic  examination  the  organism  is  seen  as 
long,  branching  threads  consisting  of  short  segments. 

It  is  non-motile.  Spore-formation  is  questionable, 
Nocard  having  seen  it,  while  Lehman  and  Neumann 
have  not.  The  organism  may  be  stained  by  the  ordi- 
nary methods,  and  also  by  the  Gram-Weigert  process. 
It  grows  on  all  the  ordinary  culture-media,  and  at  both 
room-  and  body-temperature,  especially  well  at  the 
latter.  It  is  aerobic. 

Colonies  in  agar-agar  reach  a  size  of  from  1  to  2  mm. ; 
are  yellowish  white  in  color,  irregular  in  outline,  and 
have  the  appearance  of  a  glazed,  membranous  mass. 

On  gelatin,  the  growth  is  much  slower,  so  that  after 
ten  days  the  colonies  appear  as  tiny  translucent  round 
glistening  points.  Under  low  power  of  the  micro- 
scope these  colonies  are  sharply  circumscribed,  grayish 
or  greenish  in  color,  and  are  without  characteristic 
structure. 

25 


386  BACTERIOLOGY. 

Growth  in  bouillon  is  characterized  by  a  tough,  slimy 
sediment,  and  at  times  by  more  or  less  of  pellicle-forma- 
tion. Pellicle-formation  is  encouraged  by  the  addition 
of  glycerin.  The  bouillon  is  not  uniformly  clouded  by 
the  growth. 

In  milk,  it  causes  an  alkaline  reaction,  solution  of 
casein,  but  no  coagulation. 

On  potato,  it  grows  slowly  as  a  dull  yellowish-white 
dry  membrane. 

Bovines,  sheep,  and  guinea-pigs  are  susceptible  to  in- 
oculation ;  rabbits,  dogs,  cats,  horses,  and  asses  are  not. 

When  pure  cultures  are  injected  into  either  the  circu- 
lation or  the  peritoneal  cavity  of  guinea-pigs,  death  en- 
sues in  from  nine  to  twenty  days.  The  autopsy  reveals 
diffuse  pseudotuberculosis  of  the  omentum.  Within 
the  pseudotuhercles  the  organism  is  seen  as  long, 
branching  threads,  often  matted  together  as  a  true 
mycelium. 

By  subcutaneous  inoculation  only  the  neighboring 
lymph -glands  are  affected. 

The  disease  farcin  des  boeufs  is  said  to  be  more  com- 
mon in  Guadeloupe  than  elsewhere. 

ACTINOMYCES  EppiNGERi. — This  organism  was  dis- 
covered by  Eppinger  in  an  abscess  of  the  brain.  He 
regarded  it  as  a  cladothrix,  and  gave  to  it  the  designa- 
tion cladothrix  asteroides.  It  grows  well  in  pure  cult- 
ure under  artificial  conditions,  and  is  pathogenic  for 
animals.  In  the  case  studied  by  Eppinger  the  organ- 
ism was  present  not  only  in  the  abscess,  but  also  in  the 
meninges  of  the  brain  and  cord  and  in  the  bronchial 
and  supraclavicular  lymph-glands.  There  is  no  doubt 
of  its  causal  relation  to  the  conditions. 

In  pure  culture  it  grows  well  on  ordinary  media.     It 


ACTINOMYCETES.  387 

appears  as  long,  branching  threads,  many  of  which  are 
composed  of  short  quadratic  segments.  Spores  are  not 
formed.  Motility  is  doubtful ;  it  has  been  observed  by 
Eppinger,  while  Lehman  and  Neumann  failed  to  detect 
it.  It  stains  both  by  the  ordinary  dyes  and  by  the 
method  of  Gram.  It  grows  scarcely,  if  at  all,  under 
anaerobic  conditions.  It  grows  at  room-temperature, 
but  much  better  at  the  temperature  of  the  body.  The 
best  growth  is  observed  on  nutrient  agar-agar  containing 
2  per  cent,  of  glucose.  The  colonies  on  the  surface  of 
glucose  agar-agar  appear  as  yellowish-white,  round, 
finely  granular,  dull  patches  that  are  surrounded  by  a 
narrow  paler  zone.  In  the  depths  of  the  medium  they 
do  not  develop  beyond  very  small  points. 

On  gelatin  the  growth  is  very  slow;  there  is  no 
liquefaction,  and  after  a  time  the  colonies  take  on  an 
orange-red  color. 

Bouillon  is  not  uniformly  clouded.  Growth  takes 
place  on  the  surface  in  the  form  of  a  whitish  pellicle, 
in  which  dense  white  masses  may  be  seen.  These 
latter  increase  in  size,  become  detached,  and  fall  to 
the  bottom  of  the  vessel,  to  collect  as  mycelium-like 
sediment. 

On  potato,  growth  begins  as  a  coarsely  granulated 
white  layer,  which  becomes  gradually  red  in  color.  It 
is  ultimately  covered  by  a  fine,  hair-like  growth. 

Both  rabbits  and  guinea-pigs  are  susceptible  to  its 
pathogenic  action.  When  injected  into  either  the  circu- 
lation, the  peritoneal  cavity,  or  beneath  the  skin,  there 
develop  in  from  one  to  four  weeks  a  condition  closely 
simulating  tuberculosis  ("  pseudotuberculosis  clado- 
tlirica").  The  organism  quickly  loses  its  pathogenic 
properties  under  artificial  cultivation. 


388  BA  CTERIOLOG  Y. 

ACTINOMYCES        PSEUDOTUBERCULOSIS. In       1807 

Flexner  detected  this  organism  in  a  consolidated  and 
caseous  lung.  The  condition  suggested  tuberculosis. 
The  lesion  consisted  mainly  of  an  inflammatory  exuda- 
tion that  had  undergone  caseation,  but  in  addition  there 
were  present  isolated  nodules  that  in  size  and  general 
appearance  were  difficult  to  distinguish  from  miliarv 
tubercles.  Giant  cells  were  not  seen.  The  streptothrix 
was  abundant  in  the  lung,  appearing  as  masses  of  con- 
voluted, branching  threads.  The  contours  of  the  rods 
were  not  quite  uniform,  the  staining  was  irregular,  and 
occasionally  a  thread  was  seen  that,  toward  its  extrem- 
ity, appeared  to  be  breaking  up  into  short  segments. 
No  coccus-like  forms  were  seen.  It  is  stained  best  by 
the  Weigert  method,  when  deeply  stained  masses  sepa- 
rated from  one  another  by  more  or  less  clear  spaces  are 
to  be  detected.  The  organism  was  not  obtained  in 
culture,  and  no  effect  was  produced  on  guinea-pigs 
by  subcutaneous  inoculation  with  bits  of  the  diseased 
lung.1 

1For  the  literature  on  pathogenic  streptothrices,  see  Flexner: 
Journal  of  Experimental  Medicine,  1898,  vol.  iii.  p.  435 ;  for  a  sum- 
mary of  cases  in  which  streptothrices  have  been  found,  see  Musser, 
Pearce,  and  Gwyn :  Transactions  of  the  Association  of  American 
Physicians,  1901,  vol.  xvi.  p.  208. 


CHAPTER    XVIII. 

Glanders— Characteristics  of  the  disease— Histological  structure  of 
the  glanders  nodule — Susceptibility  of  different  animals  to  glan- 
ders— The  bacterium  of  glanders  ;  its  morphological  and  cultural 
peculiarities — Diagnosis  of  glanders. 

SYNONYMS:  Eotz  (Ger.),  Morve  (Fr.). 

The  disease  is  generally  known  as  glanders  when  the 
mucous  membrane  of  the  nostrils  is  affected,  and  as  farcy 
when  the  subcutaneous  lymphatics  are  the  principal  sites 
of  involvement. 

Though  most  commonly  seen  in  the  horse  and  ass, 
glanders  is  not  rarely  met  with  in  other  animals,  and  is 
occasionally  encountered  in  man.  When  occurring 
spontaneously  in  the  horse  its  primary  seat  is  usually 
upon  the  mucous  membrane  of  the  nostrils.  It  appears 
in  the  form  of  small  gray  nodules,  about  which  the 
membrane  is  congested  and  swollen.  These  nodules 
ultimately  coalesce  to  form  ulcers.  There  is  a  profuse 
slimy  discharge  from  the  nostrils  during  the  course  of  the 
disease.  The  primary  lesion  may  extend  from  its  seat  in 
the  nose  to  the  mouth,  larynx,  trachea,  and  ultimately 
to  the  lungs.  Its  secondary  manifestations  are  observed 
along  the  lymphatics  that  communicate  with  the  initial 
focus ;  in  the  lymphatic  glands,  and  as  metastatic  foci 
in  the  internal  organs.  Less  frequently  the  disease  is 
seen  to  begin  in  the  skin,  particularly  in  the  region  of 
the  neck  and  breast.  When  in  this  locality  the  sub- 
cutaneous lymphatics  become  involved,  and  are  con- 

389 


390  BACTERIOLOGY. 

verted  into  indurated,  knotty  cords — "  farcy-buds  " — 
easily  discernible  from  without. 

In  man  it  usually  occurs  in  individuals  who  have 
been  in  attendance  upon  animals  affected  with  the  dis- 
ease. It  may  occur  upon  the  mucous  membrane  of  the 
nares ;  but  its  most  frequent  expressions  are  in  the  skin 
and  muscles,  where  appear  abscesses,  phlegmons,  ery- 
sipelas-like inflammations,  and  local  necrosis  closely 
resembling  carbuncles.  Metastases  to  the  lungs,  kid- 
neys, and  testicles,  as  in  the  horse,  may  also  be  seen. 

When  occurring  upon  the  mucous  membrane  glan- 
ders is  characterized  by  the  presence  of  gray  nod- 
ules, about  as  large  as  a  pin-head,  that  closely  re- 
semble miliary  tubercles  in  their  naked-eye  appearance. 
These  consist  histologically  of  granulation-tissue — /.  <>., 
of  small  round  cells,  very  similar  to  proliferating 
leucocytes — of  some  lymph-cells,  and,  in  the  earliest 
stages,  of  a  small  portion  of  necrotic  tissue.  As  they 
grow  older,  and  the  process  advances,  there  is  a 
tendency  to  central  necrosis,  with  the  ultimate  for- 
mation of  a  soft,  yellow,  creamy,  pus-like  material. 
Though  strikingly  like  miliary  tubercles  in  certain 
respects  in  the  early  stages,  they  present,  nevertheless, 
decided  points  of  difference  when  examined  more  in 
detail. 

The  round-cell  infiltration  of  the  glanders  nodule  con- 
sists essentially  of  polymorphonuclear  leucocytes,  while 
that  of  the  miliary  tubercle  partakes  more  of  the  nature 
of  a  lymphocytic  infiltration ;  in  the  later  stages  of  the 
process  the  glanders  nodule  breaks  down  into  a  soft, 
creamy  matter,  very  analogous  to  ordinary  pus,  while 
in  the  later  stages  of  the  miliary  tubercle  the  tendency 
is  to  an  amalgamation  of  its  histological  constituents, 


BACTERIUM  MALLEI.  391 

and  ultimately  to  necrosis  with  caseation.  The  giant- 
cell  formation  common  to  tuberculosis  is  never  seen  in 
the  glanders  nodule.  As  Baumgarten  aptly  puts  it : 
u  The  pathological  manifestations  of  glanders,  from  the 
histological  aspect,  stand  midway  between  the  acute 
purulent  and  the  chronic  inflammatory  processes." 1  JEvi- 
dently  these  differences  are  only  to  be  explained  by  differ- 
ences in  the  nature  of  the  causes  that  underlie  the  several 
affections.  We  have  studied  the  characteristics  of  bacte- 
rium tuberculosis  ;  we  shall  now  take  up  the  bacillus  of 
glanders  and  note  the  striking  differences  between  them. 

BACTERIUM    MALLEI  (LOFFLER),  MIGULA,  1900. 

Synonyms  :  Bacillus  mallei  (Loffler),  1886  ;  Eotz  bacillus,  Kranz- 
feld,  1887. 

In  1882  Loffler  and  Schiitz  discovered  in  the  dis- 
eased tissues  of  animals  suffering  from  glanders  a  bacte- 
rium that,  when  isolated  in  pure  culture  and  inoculated 
into  susceptible  animals,  possesses  the  property  of  repro- 
ducing the  disease  with  all  its  clinical  and  pathological 
manifestations.  It  is  therefore  the  cause  of  the  disease. 

This  organism  is  a  rod,  with  rounded  or  slightly 
pointed  ends.  It  usually  stains  somewhat  irregularly. 
(See  Fig.  66.)  When  examined  in  stained  prepara- 
tions its  continuity  is  marked  by  alternating  darkly 
and  lightly  stained  areas.  It  is  usually  seen  as  a 
single  rod,  but  may  occur  in  pairs,  and  less  frequently 
in  longer  filaments. 

The  question  as  to  its  spore-forming  property  is  still 
an  open  one,  though  the  weight  of  evidence  is  in  oppo- 

1  For  a  further  discussion  of  the  pathology  and  pathogenesis  of  this 
disease,  see  Lehrbuch  der  pathologischen  Mykologie,  by  Baumgarten, 
1890.  See,  also,  Wright :  "  The  Histological  Lesions  of  Acute  Glanders 
in  Man/'  Journal  of  Experimental  Medicine,  vol.  i.  p.  577. 


392 


BACTERIOLOGY. 


sition  to  the  opinion  that  it  possesses  this  peculiarity. 
Certain  observers  claim  to  have  demonstrated  spores  in 
the  bacteria  by  particular  methods  of  staining ;  but  this 
statement  can  have  but  little  weight  when  compared 
with  the  behavior  of  the  organism  when  subjected  to 
more  conclusive  tests.  For  example,  it  does  not,  at 
any  stage  of  development,  resist  exposure  to  3  per  cent. 

FIG.  66. 


Bacterium  mallei,  from  culture. 

carbolic  acid  solution  for  longer  than  five  minutes,  nor 
to  1  :  5000  sublimate  solution  for  more  than  two  min- 
utes. It  is  destroyed  in  ten  minutes  in  some  experi- 
ments, and  in  five  in  others,  by  a  temperature  of  55°  C. ; 
and  when  dried  it  loses  its  vitality,  according  to  dif- 
ferent observers,  in  from  thirty  to  forty  days ;  all  of 
which  speak  directly  against  this  being  a  spore-bearing 
bacillus. 

It    is  not   motile,    and    does   not   therefore    possess 
flagella. 


•  BACTERIUM  MALLEI.  393 

It  grows  readily  on  ordinary  nutrient  media  at  from 
25°  to  38°  C. 

Upon  nutrient  agar-agar,  both  with  and  without 
glycerin,  it  appears  as  a  moist,  opaque,  glazed  layer, 
with  nothing  characteristic  about  it.  This  is  true  both 
for  smear-cultures  and  for  single  colonies. 

Its  growth  on  gelatin  is  much  less  voluminous  than 
on  media  that  can  be  kept  at  higher  temperature,  though 
it  does  grow  on  this  media  at  room-temperature  without 
causing  liquefaction. 

Its  growth  on  blood-serum  is  in  the  form  of  a 
moist,  opaque,  slimy  layer,  inclining  to  a  yellowish  or 
dirty,  brownish-yellow  tinge.  It  does  not  liquefy  the 
serum. 

On  potato  its  growth  is  moderately  rapid,  appearing 
in  from  twenty-four  to  thirty-six  hours  at  37°  C.  as  a 
moist,  amber-yellow,  transparent  deposit  having  some- 
what the  appearance  of  honey ;  this  becomes  deeper 
in  color  and  denser  in  consistence  as  growth  progresses, 
and  finally  takes  on  a  reddish-brown  color ;  at  the  same 
time  the  potato  about  it  becomes  darkened. 

In  bouillon  it  causes  diffuse  clouding,  with  ultimately 
the  formation  of  a  more  or  less  tenacious  or  ropy  sedi- 
ment. 

In  milk  to  which  a  little  litmus  has  been  added  it 
causes  the  blue  color  to  become  red  or  reddish  in  from 
four  to  five  days,  and  quite  red  after  two  weeks  at  37°  C. 
At  the  same  time  the  milk  separates  into  clear  whey 
and  a  firm  clot  of  casein. 

Its  reactions  to  heat  are  very  interesting.  At  42°  C. 
it  will  often  grow  for  twenty  days  or  more.  It  will 
not  grow  at  43°  C.,  and  if  exposed  to  this  temperature 
for  forty-eight  hours  it  is  destroyed.  It  is  killed  in  five 


394  B  A  CTERIOL  0  G  Y. 

hours  when  exposed  to  50°  C.,  and  in  five  minutes  by 
55°  C. 

It  grows  both  with  and  without  oxygen  ;  it  is  there- 
fore facultative  as  regards  its  relation  to  this  gas. 

On  cover-slips  it  stains  readily  with  all  the  basic 
aniline  dyes,  and,  as  a  rule,  as  stated,  presents  conspic- 
uous irregularities  in  the  way  that  it  takes  up  the  dyes, 
being  usually  marked  by  deeply  stained  areas  that  alter- 
nate with  points  at  which  it  either  does  not  stain  at  all 
or  only  slightly. 

The  animals  susceptible  to  infection  by  this  organism 
are  horses,  asses,  field-mice,  guinea-pigs,  and  cats. 
Baumgarten  records  cases  of  infection  in  lions  and 
tigers  that  were  fed,  in  menageries,  with  flesh  from 
horses  affected  with  the  disease.  Rabbits  are  but 
slightly  susceptible  ;  dogs  and  sheep  still  less  so.  Man 
is  susceptible,  and  infection  not  rarely  terminates  fatally. 
White  mice,  common  gray  house-mice,  rats,  cattle,  and 
hogs  are  insusceptible. 

INOCULATION  EXPERIMENTS. — The  most  favorable 
animal  upon  which  to  study  the  pathogenic  properties 
of  this  organism  in  the  laboratory  is  the  common  field- 
mouse.  When  inoculated  subcutaneously  with  a  small 
portion  of  a  pure  culture  of  bacterium  mallei  death 
ensues  in  about  seventy-two  hours.  The  most  conspicu- 
ous tissue-changes  will  be  enlargement  of  the  spleen, 
which  is  at  the  same  time,  almost  constantly,  studded 
with  minute  gray  nodules,  the  typical  glanders  nodule. 
They  are  rarely  present  in  the  lungs,  but  may  frequently 
be  seen  in  the  liver.  From  these  nodules  the  glanders 
bacillus  may  be  obtained  in  pure  culture.  With  the 
exception  of  the  characteristic  nodules,  the  disease  as 
seen  in  this  animal  presents  none  of  the  features  that 


STAISISG   IN  TISSCES.  395 

it  displays  in  the  horse  and  ass.  The  clinical  and  path- 
ological manifestations  resulting  from  inoculation  of 
guinea-pigs  are  much  more  faithful  reproductions.  The 
animal  lives  usually  from  six  to  eight  weeks  after  inocu- 
lation, and  during  this  time  becomes  affected  with  a  group 
of  most  interesting  and  peculiar  pathological  processes. 
The  specific  inflammatory  condition  of  the  mucous 
membrane  of  the  nostrils  is  almost  always  present.  The 
joints  become  swollen  and  infiltrated  to.  such  an  extent 
as  often  to  interfere  with  the  use  of  the  legs.  In  male 
animals  the  testicles  become  enormously  distended  with 
pus,  and  on  closer  examination  a  true  orchitis  and  epi- 
didymitis  are  seen  to  be  present.  The  internal  organs, 
particularly  the  lungs,  kidneys,  spleen,  and  liver,  are 
usually  the  seat  of  the  nodular  formations  characteristic 
of  the  disease.  From  all  of  these  disease-foci  the 
bacillus  causing  them  can  be  isolated  in  pure  culture. 

STAINING  IN  TISSUES. — Though  always  present  in 
the  diseased  tissues,  considerable  trouble  is  usually 
experienced  in  demonstrating  the  bacteria  by  staining- 
methods.  The  difficulty  is  due  to  the  fact  that  the  bac- 
teria are  very  easily  decolorized,  and  in  tissues  stained 
by  the  ordinary  processes  are  robbed  of  their  color  even 
by  the  alcohol  with  which  the  tissue  is  rinsed  and  de- 
hydrated. If  we  will  remember  not  to  employ  con- 
centrated stains,  and  not  to  expose  the  sections  to  the 
stains  for  too  long  a  time,  but  little  treatment  with 
deoolorizing-agents  is  necessary,  and  very  satisfactory 
preparations  will  be  obtained.  A  number  of  methods 
have  been  snirLiysted  for  staining  the  glanders  bacilli 
in  tissues,  and  if  what  has  been  said  will  be  borne  in 
mind,  no  difficulty  should  be  experienced. 

Two  sati-t'actnrv  methods  that  we  have  used  for  this 


396  BACTERIOLOGY. 

purpose,  though  perhaps  no  better  than  some  of  the 
others,  are  as  follows  : 

a.  Transfer   the   sections    from   alcohol  to  distilled 
water.     This  lessens  the  intensity  with  which  the  stain 
subsequently  takes   hold   of  the  tissues,  by  diminish- 
ing the  activity  of  the  diffusion  that  would  occur  if 
they  were  placed  from  alcohol  into  watery  solutions  of 
the  dyes.     Transfer  from  distilled  water  to  the  slide, 
absorb   all  water  with  blotting-paper,  and   stain  with 
two  or  three  drops  of 

Carbol-fuchsin 10  c.c. 

Distilled  water 100  c.c. 

for  thirty  minutes ;  absorb  all  superfluous  stain  with 
blotting-paper,  and  wash  the  section  three  times  with 
0.3  per  cent,  acetic  acid,  not  allowing  the  acid  to  act 
for  more  than  ten  seconds  each  time.  Remove  all 
acid  from  the  section  by  carefully  washing  in  distilled 
water ;  absorb  all  water  by  gentle  pressure  with  blot- 
ting-paper ;  and  finally,  at  very  moderate  heat,  or  with 
a  small  bellows  (Ku'hne),  dry  the  section  completely  on 
the  slide.  When  dried  clear  in  xylol  and  mount  in 
xylol  balsam. 

b.  Transfer   the   sections   from   alcohol   to   distilled 
water ;  from  water  to  the  dilute  fuchsin  solution,  and 
gently    warm   (about    50°    C.)   for    fifteen   to   twenty 
minutes.     Transfer  sections  from  the  staining-solution 
to  the  slide,  absorb  all  superfluous  stain  with  blotting- 
paper,  and  then  treat  them  with  1  per  cent,  acetic  acid 
from  one-half  to  three-quarters  of  a  minute.     Remove 
all  trace  of  acid  with  distilled  water,  absorb  all  water  by 
gentle  pressure  with  blotting-paper,  and  then  treat  the 
sections  with  absolute   alcohol  by  allowing   it  to  flow 


DIAGNOSIS  BY  METHOD   OF  STEAUSS.         397 

over  them  drop  by  drop.  For  small  sections  three  or 
four  drops  are  sufficient.  Under  no  circumstances 
should  the  alcohol  be  allowed  to  act  for  more  than 
one-quarter  of  a  minute.  Clear  in  xylol  and  mount 
in  xylol  balsam. 

By  method  b  the  tissues  are  better  preserved  than  by 
method  a,  by  which  they  are  dried. 

Very  good  preparations  are  also  obtained  by  the  use 
of  Loffler's  alkaline  methylene-blue,  if  care  be  taken 
not  to  stain  for  too  long  a  time  nor  to  decolorize  too 
energetically  with  alcohol. 

No  method  of  contrast-stain  for  this  organism  in 
tissues  has  been  devised. 

In  properly  stained  tissues  the  bacteria  will  be  found 
most  numerous  in  the  centre  of  the  nodules,  becoming 
fewer  as  we  approach  the  periphery.  They  usually  lie 
between  the  cells,  but  at  times  may  be  seen  almost 
filling  some  of  the  epithelial  cells,  of  which  the  nodule 
contains  more  or  less.  They  are  always  present  in 
these  nodules  in  the  tissues ;  they  are  rarely  present 
in  the  blood,  and,  if  so,  in  only  small  numbers. 

DIAGNOSIS  OF  THE  DISEASE  BY  THE  METHOD  OF 
STRAUSS. — From  what  has  been  said,  the  diagnosis  of 
glanders  by  routine  bacteriological  methods  is  certain 
and  relatively  easy,  but  requires  time.  In  clinical  work 
it  is  of  great  importance  for  the  diagnosis  to  be  estab- 
lished as  quickly  as  possible.  With  this  in  view  Strauss 
devised  a  method  that  has  given  entirely  satisfactory 
results.  It  consists  in  introducing  into  the  peritoneal 
cavity  of  a  male  guinea-pig  a  bit  of  the  suspected  tissue 
or  culture.  If  it  be  from  a  genuine  case  of  glanders, 
the  testicles  begin  to  swell  in  about  thirty  hours,  ^nd 
as  this  proceeds  the  skin  over  them  becomes  red  and 


BACTERIOLOGY. 

shining,  desquamation  occurs,  evidences  of  pus-forma- 
tion are  seen,  and,  indeed,  the  abscess  (purulent  orchitis) 
often  breaks  through  the  skin.  The  diagnostic  sign  is 
the  tumefaction  of  the  testicles. 

MALLEIN. — The  filtered  products  of  growth  of  the 
glanders  bacillus  in  fluid  media  represent  what  is  known 
as  mallein — a  solution  of  compounds  that  bear  to 
glanders  a  relation  analogous  to  that  which  tuberculin 
bears  to  tuberculosis.  It  is  used  with  considerable  suc- 
cess as  a  diagnostic  aid  in  detecting  the  existence  or 
absence  of  deep-seated  manifestations  of  the  disease,  the 
glanderous  animal  reacting  (manifested  by  elevations  of 
body -temperature  greater  than  1.5°  C.)  to  subcutaneous 
injections  of  mallein  in  from  four  to  ten  hours,  while  an 
animal  not  so  affected  gives  no  such  reactions. 

Mallein  is  prepared  from  old  glycerin-bouillon  cult- 
ures of  the  glanders  bacterium  by  steaming  them  for 
several  hours  in  the  sterilizer,  after  which  they  are  fil- 
tered through  unglazed  porcelain. 

By  some  it  is  said  that  the  repeated  injection  of  mal- 
lein in  small  doses  confers  immunity  from  infection  by 
bacterium  mallei  upon  animals  so  treated ;  an  opinion 
that  is  entirely  in  accord  with  the  principles  underlying 
the  artificial  induction  of  immunity  in  general. 


CHAPTER    XIX. 

Bacterium  (syn.  Bacillus)  diphtlterise — Its  isolation  and  cultivation — 
Morphological  and  cultural  peculiarities — Pathogenic  properties  — 
Variations  in  virulence — Bacterium  pseudodiphtheriticiim — Bacte- 
rium xerosis — Diphtheria  antitoxin. 

FROM  the  gray-white  deposit  on  the  fauces  of  a  diph- 
theritic patient  prepare  a  series  of  cultures  in  the  fol- 
lowing way  : 

Have  at  hand  five  or  six  tubes  of  Loffler's  blood- 
serum  mixture.  (See  chapter  on  Media.)  Pass  a  stout 
platinum  needle,  which  has  been  sterilized,  into  the 
membrane  and  twist  it  around  once  or  twice,  or  brush 
it  gently  over  the  surface  of  the  membrane.  Without 
touching  it  against  anything  else  rub  it  carefully  over 
the  surface  of  one  of  the  serum-tubes ;  without  steriliz- 
ing it  pass  it  over  the  surface  of  the  second,  then  the 
third,  fourth,  and  fifth  tubes.  Place  these  tubes  in  the 
incubator.  Then  prepare  cover-slips  from  scrapings 
from  the  membrane  on  the  fauces.  If  the  case  is  one 
of  true  diphtheria,  the  tubes  will  be  ready  for  examina- 
tion on  the  following  day. 

The  reason  that  plates  are  not  made  in  the  regular 
way  in  this  examination  is  that  the  bacillus  of  diph- 
theria develops  much  more  luxuriantly  on  the  serum 
mixture,  from  which  plates  cannot  be  made,  than  it 
does  on  the  media  from  which  they  can  be  made.  The 
method  employed,  however,  insures  a  dilution  in  the 
number  of  organisms  present,  and  this,  in  addition  to 
the  fact  that  the  blood-serum  mixture  is  a  much  more 
favorable  medium  for  the  rapid  development  of  the 
diphtheria  organism  than  of  the  other  organisms  present, 

399 


400  BACTERIOLOGY. 

makes  its  isolation  by  this  method  a  matter  of  but  little 
difficulty. 

After  twenty-four  hours  in  the  incubator  the  tubes 
present  a  characteristic  appearance.  Their  surfaces  are 
marked  by  more  or  less  irregular  patches  of  a  white 
or  cream-colored  growth,  which  is  usually  more  dense 
at  the  centre  than  at  the  periphery.  Except  now  and 
then,  when  a  few  orange-colored  colonies  may  be  seen, 
these  large  irregular  patches  are  the  most  conspicuous 
objects  on  the  surface  of  the  serum.  Occasionally, 
almost  nothing  else  appears. 

The  cover-slips  made  from  the  membrane  at  the  time 
the  cultures  were  prepared  will  be  found  on  microscopic 
examination  to  present,  in  many  cases,  a  great  variety  of 
organisms;  but  conspicuous  among  them  will  be  noticed 
slightly  curved  bacilli  of  irregular  size  and  outline. 
In  some  cases  they  will  be  more  or  less  clubbed  at 
one  or  both  ends;  sometimes  they  appear  spindle  in 
shape,  again  as  curved  wedges;  now  and  then  they  are 
irregularly  segmented.  They  are  rarely  or  never  regu- 
lar in  outline.  If  the  preparation  has  been  stained 
with  Loffler's  alkaline  methylene-blue  solution,  many 
of  these  irregular  rods  are  seen  to  be  marked  by  cir- 
cumscribed points  in  their  protoplasm  which  stain  very 
intensely — they  appear  almost  black.  This  irregularity 
in  outline  is  the  morphological  characteristic  of  bacillus 
diphtherice  of  Loffler. 

It  must  be  remembered,  however,  that  the  diagnosis 
of  diphtheria  should  not  under  all  circumstances  be 
made  from  the  examination  of  cover-slip  preparations 
alone,  especially  when  they  are  stained  only  by  the 
usual  method  —  i.  e.,  with  Loffler's  methylene-blue. 
There  are  other  organisms  present  in  the  mouth-cavity, 
particularly  in  the  mouths  of  persons  having  decayed 


BACTERIUM  DIPHTHERIA.  401 

teeth,  the  morphology  of  which  is  so  like  that  of  the 
bacillus  of  diphtheria  that  they  might  easily  be  mistaken 
for  that  organism  if  subjected  to  only  the  usual  method 
of  microscopic  examination ;  and  again,  the  genuine 
diphtheria  organism  is  sometimes  found  in  the  mouth- 
cavities  of  healthy  persons  in  attendance  upon  diph- 
theria cases,  such  persons  being  at  the  time  insusceptible 
to  the  pathogenic  activities  of  the  organism.  In  the 
vast  majority  of  instances,  however,  where  the  clinical 
condition  of  the  patient  justifies  a  suspicion  of  diph- 
theria, a  microscopic  examination  alone  of  the  deposit 
in  the  throat  will  serve  to  confirm  or  contradict  this 
opinion,  and  such  examinations  very  frequently  reveal 
the  diphtheritic  nature,  etiologically  speaking,  of  mild 
conditions  of  the  throat  which  are  not  associated  with 
grave  constitutional  manifestations. 

BACTERIUM    DIPHTHERIA    (LOFFLER),    MIGULA,  1900. 

Synonyms:  Bacillus  diphtherias,  Loffler,  1884;  Klebs-Loffler  bacil- 
lus ;  Corynebacterium  diphtherise,  Lehmann  and  Neumann.  1896. 

Bacterium  diphtheria?,  discovered  microscopically  by 
Klebs,  and  isolated  in  pure  culture  and  proved  to 
stand  in  causal  relation  to  diphtheria  by  Loffler,  can 
readily  be  identified  by  its  cultural  peculiarities  and 
by  its  pathogenic  activity  when  introduced  into  tissues 
of  susceptible  animals.  In  guinea-pigs  and  kittens  the 
results  of  its  growth  are  histologically  identical  with 
those  found  in  the  bodies  of  human  beings  who  have 
died  of  diphtheria. 

When  studied  in  pure  culture  its  morphological  and 
cultural  peculiarities  are  as  follows : 

MORPHOLOGY. — As  obtained  directly  from  the  diph- 
theritic deposit  in  the  throat  of  an  individual  sick  of 
the  disease,  it  is  sometimes  comparatively  regular  in 

26 


402  BA  CTERIOLOG  Y. 

shape,  appearing  as  straight  or  slightly  curved  rods  with 
more  or  less  pointed  ends.  More  frequently,  however, 
spindle-  and  club-shapes  occur,  and  not  rarely  many  of 
these  rods  stain  irregularly ;  in  some  of  them  very 
deeply  stained  round  or  oval  points  can  be  detected. 

When  cultures  are  examined  microscopically  it  is 
especially  characteristic  to  find  irregular,  bizarre  forms, 
such  as  rods  with  one  or  both  ends  swollen,  and  very 
frequently  rods  broken  at  irregular  intervals  into  short, 
sharply  defined  segments,  either  round,  oval,  or  with 
straight  sides.  Some  forms  stain  uniformly,  others  in 
various  irregular  ways,  the  most  common  being  the 
appearance  of  deeply  stained  granules  in  a  lightly 
stained  bacillus. 

By  a  series  of  studies  upon  this  organism  when  cul- 
tivated under  artificial  conditions  we  have  found  that 
its  form  and  size  depend  very  largely  upon  the  nature 
of  its  environment.  That  is  to  say,  its  morphology  is 
always  more  regular,  and  it  is  smaller  on  glycerin-agar- 
agar  than  on  other  media  used  for  its  cultivation  ;  while 
upon  Loffler's  blood-serum  the  other  extremes  of  de- 
velopment appear :  here  one  sees,  instead  of  the  very 
short,  spindle,-  lancet,-  club-shaped,  always  segmented 
and  regularly  staining  forms  as  seen  upon  glycerin-agar- 
agar,  long,  sometimes  extremely  slender,  sometimes 
thicker,  irregularly  staining  threads  that  are  usually 
clubbed  and  frequently  pointed  at  their  extremities. 
They  are,  as  a  rule,  marked  by  areas  that  stain  more 
intensely  than  does  the  rest  of  the  rod,  and  at  times  they 
may  be  a  little  swollen  at  the  centre.  These  differences 
are  so  conspicuous  that  microscopic  preparations  from 
cultures  from  the  same  source,  but  cultivated  in  the  one 
case  on  glycerin-agar-agar  and  in  the  other  upon  blood- 


BACTERIUM  DIPHTHERIA.  403 

serum,  when  placed  side  by  side  would  hardly  be  recog- 
nized as  of  the  same  organism,  unless  its  peculiar  be- 
havior under  these  circumstances  was  already  known. 
Another  peculiar  variation  is  that  observed  upon  very 
slightly  acid  blood-serum.  Here  the  rods  appear 
swollen,  and  are  usually  contracted  to  oval  or  short, 
oblong  bodies,  which  stain  very  faintly,  and  in  which 
are  usually  located  one  or  two  very  deeply  staining 
round  or  oval  points.  Various  authors  have  called 
attention  to  branching  forms  of  this  organism  that  are 
occasionally  encountered,  especially  when  cultivated 
upon  albumin.  We  have  never  seen  the  branching  diph- 
theria organisms  under  conditions  that  might  reasona- 
bly be  regarded  as  favorable  to  normal  development ; 
and  in  many  thousand  blood-serum  cultures  from  cases 
of  diphtheria  that  have  been  examined  by  competent 
bacteriologists  at  the  laboratory  of  the  Board  of  Health 
of  Philadelphia,  the  branching  forms  of  this  organism 
have  not  been  observed  in  a  single  instance.  It  is  fair 
to  assume,  therefore,  that  this  peculiar  morphological 
variation  of  bacillus  diphtherice  is,  under  normal  condi- 
tions of  growth,  comparatively  rare. 

On  the  other  hand,  if  the  organism  be  grown  on 
media  favorable  to  involution,  such,  for  instance,  as  hard- 
boiled  egg,  or  coagulated  egg  of  acid  reaction,  branching 
may  be  seen,  but  with  it  degenerated  organisms  are  so 
conspicuous  as  to  leave  no  doubt  that  the  so-called 
branching  and  involution  are  attributable  to  the  same 
cause,  namely,  unsuitable  conditions  of  cultivation. 

On  plain  nutrient  agar-agar  (that  is,  nutrient  agar- 
agar  without  glycerin)  ;  on  a  medium  consisting  of  dried 
commercial  albumin  dissolved  in  bouillon  (about  10 
grammes  of  albumin  to  100  c.c.  of  bouillon  containing 


404 


BACTERIOLOGY. 


1  per  cent,  of  grape-sugar) ;  in  bouillon  without  glycerin, 
and  in  bouillon  to  which  a  bit  of  hard-boiled  egg  has 
been  added,  the  morphology  of  the  organism  is  about  in- 
termediate, in  both  size  and  outline,  between  the  forms 
seen  upon  glycerin  agar-agar  and  upon  Loffler's  blood- 
serum.  There  will  appear  about  an  equal  number  of 
short  segmented  and  longer,  irregularly  staining  forms ; 
but  in  general  the  longest  are  rarely  as  long  as  the  long 
forms  seen  on  blood-serum,  and  throughout  they  are  not 
so  conspicuous  for  the  irregularity  of  their  staining. 

FIG.  67. 


Bacterium  diph'herix.  A.  Its  morphology  on  glycerin-agar-agar.  B.  Its 
morphology  on  Loffler's  blood-serum,  c.  Its  morphology  on  acid  blood- 
serum  mixture. 

In  cultures  made  upon  two  sets  of  nutrient  agar-agar 
tubes,  differing  only  in  the  fact  that  one  set  contains 
glycerin  to  the  extent  of  6  per  cent.,  while  the  other  set 
contains  none,  a  noticeable  difference  in  morphology  can 


BACTERIUM  DIPHTHERIA.  405 

usually  be  made  out :  while  the  forms  on  the  glycerin- 
agar-agar  cultures  are  throughout  small,  and  pretty 
regular  in  size,  shape,  and  staining,  those  on  the  plain 
agar-agar  are  larger,  stain  less  uniformly,  vary  more  in 
shape,  and  when  stained  by  Loffler's  blue  are  not  so 
regularly  marked  by  pale  transverse  lines  that  give  to 
them  the  appearance  of  being  made  up  of  numerous 
short  segments. 

Though  the  outline  of  this  organism  is  more  regular 
under  some  circumstances  than  others,  it  is  nevertheless 
always  conspicuous  for  its  manifold  variations  in  shape. 

GROWTH  ON  SERUM  MIXTURE. — The  medium  upon 
which  bacillus  diphtheria  grows  most  rapidly  and  lux- 
uriantly, and  which  is  best  adapted  for  determining  its 
presence  in  diphtheritic  exudates,  is,  as  has  been  stated, 
the  blood-serum  mixture  of  Loffler.  (See  chapter  on 
Media.)  On  the  blood-serum  mixture  the  colonies  of 
bacillus  diphtheria?  grow  so  much  more  rapidly  than  the 
other  organisms  usually  present  in  secretions  and  exuda- 
tions in  the  throat  that  at  the  end  of  twenty-four  hours 
they  are  often  the  only  colonies  that  attract  attention  ;  and 
if  others  of  similar  size  are  present,  they  are  generally  of 
quite  a  different  aspect.  Its  colonies  are  large,  round, 
elevated,  grayish-white  or  yellowish,  wTith  a  centre  more 
opaque  than  the  slightly  irregular  periphery.  The  sur- 
face of  the  colony  is  at  first  moist,  but  after  a  day  or 
two  becomes  rather  dry  in  appearance. 

A  blood-serum  tube  studded  with  coalescent  or  scat- 
tered colonies  of  this  organism  is  so  characteristic  that 
one  familiar  with  the  appearance  can  anticipate  with  tol- 
erable certainty  the  results  of  microscopic  examination. 

GLYCERIN  AGAR-AGAR. — Upon  nutrient  glycerin 
agar-agar  the  colonies  likewise  present  an  appearance 


406  XAUTERrOLOGY. 

that  may  readily  be  recognized.  They  are  in  every 
way  more  delicate  in  structure  than  when  on  the  serum 
mixture.  They  appear  at  first,  when  on  the  sur- 
face, as  very  flat,  almost  transparent,  dry,  non-glisten- 
ing, round  points  which  are  not  elevated  above  the 
surface  upon  which  they  are  growing.  When  slightly 
magnified  they  are  seen  to  be  granular,  and  to  present 
an  irregular  central  marking,  which  is  denser  and  darker 
by  transmitted  light  than  the  thin,  delicate  zone  which 
surrounds  it.  As  the  colony  increases  in  size  the  thin 
granular  peripheral  zone  becomes  broader,  is  usually 
marked  by  ridges  or  cracks,  and  its  periphery  is  notched 

FIG.  68. 


CM- 


Colonies  of  bacterium  diphtherix  on  glyccrin-a,gnr-agar.  a.  Colonies  located 
in  the  depths  of  the  medium,  b.  Colonies  just  breaking  out  upon  the  sur- 
face of  the  medium,  c.  Fully  developed  surface-colony. 

or  scalloped.  (Fig.  68,  c.)  These  colonies  are  always 
quite  dry  in  appearance.  When  dee])  down  in  the  agar- 
agar  they  are  coarsely  granular.  (Fig.  68,  a.)  They 
rarely  exceed  3  mm.  in  diameter. 

GELATIN. — On  gelatin  the  colonies  develop  much 
more  slowly  than  on  media  that  can  be  retained  at  a 
higher  temperature.  They  rarely  present  their  char- 
acteristic appearances  on  gelatin  in  less  than  seventy- 
two  hours.  They  then  appear  as  flat,  dry,  translucent 
points,  usually  round  in  outline. 


BACTERIUM  DIPHTHERIJE.  407 

When  magnified  slightly  the  centre  is  seen  to  be  more 
dense  than  the  surrounding  zone  or  zones,  for  they  are 
sometimes  marked  by  a  concentric  arrangement  of 
/ones.  The  periphery  is  irregularly  notched.  Like 
the  colonies  seen  on  agar-agar,  they  are  granular,  but 
are  much  more  granular  when  seen  in  the  depths  of  the 
gelatin  than  when  on  its  surface.  On  gelatin  the  col- 
onies rarely  become  very  large;  usually  they  do  not 
exceed  1.5  mm.  in  diameter. 

BOUILLON. — In  bouillon  it  usually  grows  in  fine 
clumps,  which  fall  to  the  bottom  of  the  tube,  or  become 
deposited  on  its  sides  without  causing  diffuse  clouding 
of  the  bouillon.  Sometimes  there  are  exceptions  to 
this  naked-eye  appearance  :  the  bouillon  may  be  dif- 
fusely clouded ;  but  if  one  inspect  it  very  closely,  par- 
ticularly if  he  examine  it  microscopically  as  a  hanging 
drop,  the  arrangement  in  clumps  will  always  be  de- 
tected, but  the  clumps  are  so  small  as  not  to  be  dis- 
cernible by  the  unaided  eye. 

In  bouillon  kept  at  a  temperature  of  35°-37°  C.  a 
soft,  whitish  pellicle  often  forms  upon  the  surface. 

Changes  in  reactions  of  the  bouillon.  The  reaction  of 
the  bouillon  frequently  becomes  at  first  acid,  and  sub- 
sequently again  alkaline,  changes  which  can  be  observed 
in  cultivations  in  bouillon  to  which  a  little  rosolic  acid 
has  been  added.  This  play  of  reactions  has  been  attrib- 
uted to  the  primary  fermentation  of  the  muscle-sugar 
often  present  in  the  bouillon.  It  does  not  occur  when 
the  medium  is  free  from  carbohydrates. 

POTATO. — On  potato  at  a  temperature  of  35°-37°  C. 
its  growth  after  several  days  is  invisible,  only  a  thinr 
dry  glaze  appearing  at  the  point  at  which  the  potato 
was  inoculated.  Microscopic  examination  of  scrapings 


408  BACTERIOLOG  Y. 

from  the  potato,  after  twenty-four  hours  at  35°-37°  0., 
reveals  a  decided  increase  in  the  number  of  individual 
organisms  planted. 

STAB-  AND  SLANT-CULTURES. — In  stab-  and  slant- 
cultures  on  both  gelatin  and  glycerin  agar-agar  the  sur- 
face-growth is  seen  to  predominate  over  that  along  the 
track  of  the  needle  in  the  depths  of  the  media. 

Isolated  colonies  on  the  surface  of  either  of  the  media 
in  this  method  of  cultivation  present  the  same  charac- 
teristics that  have  been  given  for  the  colonies  on  plates. 

The  growth  in  simple  stab-cultures  does  not  extend 
laterally  very  far  beyond  the  point  at  which  the  needle 
entered  the  medium. 

It  is  a  non-motile  organism. 

It  does  not  form  spores. 

It  is  killed  in  ten  minutes  by  a  temperature  of  58°  C. 

It  grows  at  temperatures  ranging  from  22°  to  37° 
C.,  but  most  luxuriantly  at  the  latter  temperature. 

Its  growth  in  the  presence  of  oxygen  is  more  active 
than  when  this  gas  is  excluded. 

STAINING. — In  cover-slip  preparations  made  either 
from  the  fauces  of  a  diphtheritic  patient  or  from  a  pure 
culture  of  the  organism  it  is  seen  to  stain  readily  with 
the  ordinary  aniline  dyes.  It  stains  also  by  the  method 
of  Gram,  but  the  best  results  are  obtained  by  the  use 
of  Loffler's  alkaline  methylene-blue  solution  ;  this  brings 
out  the  dark  points  in  the  protoplasmic  body  of  the 
bacilli  and  thus  aids  in  their  identification. 

For  the  purpose  of  demonstrating  the  Loffler  bacil- 
lus in  sections  of  diphtheritic  membrane,  both  the  Gram 
method  and  the  fibrin  method  of  Weigert  give  excellent 
results. 

PATHOGENIC  PROPERTIES. — When   inoculated   sub- 


BACTERIUM  1)I1>HTHERIM  409 

cutaneously  into  the  bodies  of  susceptible  animals  the 
result  is  not  the  production  of  septicaemia,  as  is  seen  to 
follow  the  introduction  into  animals  of  certain  other 
organisms  with  which  we  shall  have  to  deal,  but  the 
bacillus  of  diphtheria  remains  localized  at  the  point 
of  inoculation,  rarely  disseminating  further  than  the 
nearest  lymphatic  glands.  It  develops  at  the  point  in 
the  tissues  at  which  it  is  deposited,  and  during  its  de- 
velopment gives  rise  to  changes  in  the  tissues  which 
result  entirely  from  the  absorption  of  poisonous  albu- 
mins generated  by  the  bacteria  in  the  course  of  their 
development. 

In  a  certain  number  of  cases l  diphtheria  bacilli  have 
been  found  in  the  blood  and  internal  organs  of  individ- 
uals dead  of  the  disease  ;  but  all  that  has  been  learned 
from  careful  study  of  the  secondary  manifestations  of 
diphtheria  tends  to  the  opinion  that  they  are  in  no  way 
dependent  upon  the  immediate  presence  of  bacteria,  and 
that  the  occasional  appearance  of  diphtheria  bacteria  in 
the  internal  organs  is  in  all  probability  accidental,  and 
usually  unimportant. 

By  special  methods  of  inoculation2  (the  injection  of 
fluid  cultures  into  the  testicles  of  guinea-pigs)  diph- 
theria bacilli  can  be  caused  to  appear  in  the  ornentum  ; 
but  this  is  purely  an  artificial  manifestation  of  the  dis- 
ease, and  one  that  is  probably  never  encountered  in  the 
natural  course  of  events.  More  rarely  similar  results 
follow  upon  subcutaneous  inoculation. 

1  Frosch  :  "Die  Verbreitung  des  Diphtheric-bacillus  in  Korper  des 
Menschen,"  Zeit.   fur  Hygiene   und  Infektionskrankheiten,  1893,  Bd. 
xiii.  pp.  49-52.     Booker :  Archives  of  Pediatrics,  Aug.  1893.    Wright 
and  Stokes:  Boston  Med.  and  Surg.  Journ.,  March  and  April,  1895. 

2  Abbott  and  Ghriskey  :  "  A  Contribution  to  the  Pathology  of  Experi- 
mental Diphtheria,"  The  Johns  Hopkins  Hospital  Bulletin,   No.  30, 
April,  1893. 


410  BACTERIOLOGY. 

If  a  very  minute  portion  of  either  a  solid  or  fluid 
pure  culture  of  this  organism  be  introduced  into  the 
subcutaneous  tissues  of  a  guinea-pig  or  kitten,  death  of 
the  animal  ensues  in  from  twenty-four  hours  to  five 
days.  The  usual  changes  are  an  extensive  local  oedema, 
with  more  or  less  hypersemia  and  ecchymoses  at  the 
site  of  inoculation  ;  swollen  and  reddened  lymphatic 
glands  ;  increased  serous  fluid  in  the  peritoneum,  pleura, 
and  pericardium ;  enlarged  and  hemorrhagic  adrenal 
bodies  ;  occasionally  slightly  swollen  spleen  ;  and  some- 
times fatty  degeneration  in  the  liver,  kidney,  and  myo- 
cardium. In  guinea-pigs,  especially,  the  liver  often 
shows  numerous  macroscopic  dots  and  lines  on  the  sur- 
face and  penetrating  the  substance  of  the  organ.  They 
vary  in  size  from  a  pin-point  to  a  pin-head,  and  may  be 
even  larger.  They  are  white  and  do  not  project  above 
the  surface  of  the  capsule. 

The  bacteria  are  always  to  be  found  at  the  site  of 
inoculation,  most  abundant  in  the  grayish-white,,  fibrino- 
purulent  exudate.  They  become  fewrer  at  a  distance 
from  this,  so  that  the  more  remote  parts  of  the  cedema- 
tous  tissues  do  not  contain  them.  They  are  found  not 
only  free,  but  contained  in  large  number  in  leucocytes, 
some  of  which  have  fragmented  nuclei,  or  have  lost 
their  nuclei.  The  bacteria  within  leucocytes,  as  well  as 
some  outside,  frequently  stain  very  faintly  and  irregu- 
larly, and  may  appear  disintegrated  and  dead. 

Culture-tubes  inoculated  from  the  blood,  spleen,  liver, 
kidneys,  adrenal  bodies,  distant  lymphatic  glands,  and 
serous  transudates,  generally  yield  negative  results  ;  and 
negative  results  are  also  obtained  when  these  organs  are  { 
examined  microscopically  for  the  bacteria. 

Microscopic  examination  of  the  tissues  at  the  site  of 


BACTERIUM  DIPHTHERIA.  411 

inoculation,  as  well  as  of  the  liver,  spleen,  kidneys, 
lymphatic  glands,  and  elsewhere,  reveals  the  presence 
of  localized  foci  of  cell-death,  characterized  by  a  pecu- 
liar fragmentation  of  the  nuclei  of  the  cells  of  these 
parts. 

This  destruction  of  nuclei  results  in  the  formation 
of  groups  of  irregularly  shaped,  deeply  staining  bodies, 
having  at  times  the  appearance  of  particles  of  dust, 
while  again  they  may  be  much  larger.  Some  of  them 
are  tolerably  regular  in  outline,  while  others  are  irregu- 
larly crescentic,  dumb-bell,  flask-shape,  whetstone- 
shape,  or  bladder-like  in  form.  Occasionally  nuclei 
having  the  appearance  of  being  pinched  or  drawn  out 
can  be  seen.  At  some  points  the  fragments  are  grouped 
in  isolated  masses,  indicating  the  location  of  the 
nucleus  from  the  destruction  of  which  they  originated. 
These  particles  always  stain  much  more  intensely  than 
do  the  normal  nuclei  of  the  part.1  Oertel  showed  long 
ago  that  these  peculiar  alterations  in  their  distribution 
are  characteristic  of  human  diphtheria,  and  the  demon- 
stration of  similar  changes  in  animals  inoculated  with 
this  organism  is  important  additional  proof  that  diph- 
theria is  caused  by  it. 

By  the  inoculation  of  certain  animals  an  affec- 
tion may  be  produced  in  all  respects  identical  with 
diphtheria  as  it  exists  in  man.  If  one  open  the 
trachea  of  a  kitten  and  rub  upon  the  mucous  mem- 
brane a  small  portion  of  a  pure  culture  of  this  organ- 
ism, the  death  of  the  animal  usually  ensues  in  from  two 

1  See  "The  Histological  Changes  in  Experimental  Diphtheria,"  also 
"TlK>  Histological  Lesions  Produced  by  the  Toxalbumin  of  Diph- 
theria," by  Welch  and  Flexner,  Johns  Hopkins  Hospital  Bulletin, 
August,  1891,  and  March,  1892. 


412  BACTERIOLOGY. 

to  four  days.  At  autopsy  the  wound  will  be  found 
covered  with  a  grayish,  adherent,  necrotic,  distinctly 
diphtheritic  layer.  Around  the  wound  the  subcuta- 
neous tissues  will  be  oedematous.  The  lymphatic  glands 
at  the  angle  of  the  jaws  will  be  swollen  and  reddened. 
The  mucous  membrane  of  the  trachea  at  the  point  upon 
which  the  bacteria  were  deposited  will  be  covered  with 
a  tolerably  firm,  grayish-Avhite,  loosely  attached  pseudo- 
membrane  in  all  respects  identical  with  the  croupous 
membrane  observed  in  the  same  situation  in  cases  of 
human  diphtheria.  In  the  pseudo-membrane  and  in 
the  O3dematous  fluid  about  the  skin-wound  bacillus 
diphtheria  may  be  found  both  in  cover-slips  and  in 
cultures. 

From  what  we  have  seen — the  localization  of  the 
bacilli  at  the  point  of  inoculation,  their  absence  from 
the  internal  organs,  and  the  changes  brought  about  in 
the  cellular  elements  of  the  internal  organs — there  is 
but  one  interpretation  for  this  process,  viz.,  that  it  is 
due  to  the  production  of  a  soluble  poison  by  the  bac- 
teria growing  at  the  site  of  inoculation,  which,  gaining 
access  to  the  circulation,  produces  the  changes  that  we 
observe  in  the  tissues  of  the  internal  viscera. 

This  poison  has  been  isolated  from  cultures  of  ba- 
cillus diphtherice,  and  is  found  to  belong,  not  to  the 
crystallizable  ptomaines,  but  to  the  toxic  albumins — 
bodies  which,  in  their  chemical  composition,  are  analo- 
gous to  the  poison  of  certain  venomous  serpents.  By 
the  introduction  of  this  toxalbumin,  as  it  is  called,  into 
the  tissues  of  guinea-pigs  and  rabbits  the  same  patho- 
logical alterations  may  be  produced  that  we  have  seen 
to  follow  inoculation  with  the  bacilli  themselves,  except, 
perhaps,  the  production  of  false  membranes. 


BACTERIUM  DIPHTHERIA.  413 

Under  certain  circumstances  with  which  we  are 
not  acquainted  bacillus  diphtherice  becomes  diminished 
in  virulence  or  may  lose  it  entirely,  so  that  it  is  no 
longer  capable  of  producing  death  of  susceptible  ani- 
mals, and  may  cause  only  a  transient  local  reaction 
from  which  the  animal  entirely  recovers.  Sometimes 
this  reaction  is  so  slight  as  to  be  overlooked,  and 
again  careful  search  may  fail  to  reveal  evidence  of 
any  reaction  at  all.  This  exhibition  of  the  extremes  of 
its  pathogenic  properties,  viz.,  death  of  the  animal,  on 
the  one  hand,  and  only  very  slight  local  effects  on  the 
other,  was  at  one  time  thought  to  indicate  the  existence 
of  two  separate  and  distinct  organisms  that  were  alike 
in  cultural  and  morphological  peculiarities,  but  which 
differed  in  their  disease-producing  power.  Further 
studies  on  this  point  have,  however,  shown  that  the 
genuine  bacillus  diphtherias  may  possess  almost  all 
grades  of  virulence,  and  that  absence  of  or  dimi- 
nution in  virulence  can  hardly  serve  to  distinguish 
as  separate  species  those  varieties  that  are  otherwise 
alike ;  moreover,  the  histological  conditions  found  at  the 
site  of  inoculation  in  animals  that  have  not  succumbed, 
but  in  which  only  the  local  reaction  has  appeared,  are 
in  most  cases  characterized  by  the  same  changes  that 
are  seen  at  autopsy  in  animals  in  which  inoculation  has 
proved  fatal. 

In  the  course  of  their  observations  upon  a  large 
number  of  cases  Roux  and  Yersin  found  that  it  was 
not  difficult  to  detect,  in  the  diphtheritic  deposits  of 
the  same  individual,  bacteria  of  identical  cultural  and 
morphological  peculiarities,  but  of  very  different  degrees 
of  virulence,  and  that  with  the  progress  of  the  disease 


414  £A  CTERIOLO&  Y. 

toward  recovery  the  less  virulent  varieties  often  became 
quite  frequent.1 

There  is,  moreover,  a  mild  form  of  diphtheria,  etio- 
logically  speaking,  affecting  only  the  mucous  membrane 
of  the  nares,  known  as  membranous  rhinitis,  from 
which  it  is  very  common  to  obtain  cultures  in  all  re- 
spects identical  with  those  from  typical  diphtheria,  save 
for  their  inability  to  kill  susceptible  animals.  On  inocu- 
lation these  cultures  produce  only  local  reactions,  but 
these  are  characterized  histologically  by  the  same  kind 
of  tissue-changes  that  follow  inoculation  with  the  fully 
virulent  organism. 

Clinically,  membranous  rhinitis  is  never  such  an 
alarming  disease  as  is  laryngeal  or  pharyngeal  diph- 
theria, and,  as  stated,  the  organisms  causing  it  are  often 
of  a  low  degree  of  virulence,  though  they  are,  never- 
theless, genuine  diphtheria  bacteria. 

For  those  organisms  that  are  in  all  respects  identical 
with  the  virulent  bacillus  diphtherice,  save  for  their  ina- 
bility to  kill  guinea-pigs,  the  designation  "  pseudo-diph- 
theritic bacillus  "  is  usually  employed ;  but  from  such 
observations  as  those  just  cited  we  are  inclined  to  the 
opinion  that  psemfo-diphtheritic,  as  applied  to  an  organ- 
ism in  all  respects  identical  with  the  genuine  bacterium, 
except  that  it  is  not  fatal  to  susceptible  animals,  is  a 
misnomer,  and  that  it  would  be  more  nearly  correct  to 
designate  this  organism  as  the  attenuated  or  non-viru- 
lent diphtheritic  bacterium,  reserving  the  term  "  pseudo- 
diphtheritic  "  for  that  organism  or  group  of  organisms 
(for  there  are  probably  several)  that  is  enough  like  the 

1  It  must  not  be  assumed  from  this  that  the  bacteria  lose  their  viru- 
lence entirely,  or  that  they  all  become  attenuated  with  the  establish- 
ment of  convalescence,  for  this  is  contrary  to  what  experience  has 
shown  to  be  the  case. 


BACTERIUM  PSEUDODIPHTHER1T1CUM.       415 

diphtheria  bacterium  to  attract  attention,  but  is  distin- 
guishable from  it  by  certain  morphological  and  cultural 
peculiarities  aside  from  the  question  of  virulence. 

It  is  a  well-known  fact  that  many  pathogenic  organ- 
isms — conspicuous  among  these  being  bacterium  pneu- 
m,onicej  micrococcus  aureuSj  streptococcus  pyogenes,  and 
the  group  of  so-called  "  hemorrhagic  septicaemia " 
organisms — undergo  marked  variations  in  their  patho- 
genic properties ;  and  yet  these  organisms,  when  found 
either  devoid  of  this  peculiarity,  or  possessing  it  in  a 
diminished  degree,  are  not  designated  as  "  pseudo " 
forms,  but  simply  as  the  organisms  themselves,  the  viru- 
lence of  which,  from  various  causes,  has  been  modified. 

It  must  nevertheless  be  admitted  that  in  the  course 
of  microscopic  examination  of  materials  from  various 
sources,  including  the  pharynx,  one  occasionally  encoun- 
ters micro-organisms  whose  morphology  is  so  like  that 
of  the  genuine  bacterium  dlphtherm  as  to  create  suspi- 
cion, and  yet  they  are  at  the  same  time  sufficiently 
unlike  it  to  make  one  cautious  in  forming  an  opinion 
as  to  their  real  nature. 

BACTERIUM  PSEUDODIPHTHERITICUM. — For  a  long 
time  bacterium  pseudodiphtheriticum  was  looked  upon  as 
being  entirely  harmless,  and  the  only  particular  in  which 
it  was  regarded  as  being  of  importance  was  in  the  fact 
that  ic  was  likely  to  be  mistaken  for  bacterium  diph- 
theriae.  The  wide  dissemination  of  this  class  of  organ- 
isms and  the  demonstration  of  pathogenic  effects  in  iso- 
lated instances  has  led  to  the  more  systematic  study  of 
members  of  this  group  of  organisms. 

Bacterium  pseudodipht/ieriticum,  as  found  under  dif- 
ferent conditions,  varies  markedly  in  its  morphologic 
and  biologic  characters.  Some  of  the  varieties  have 


416  BA  CTERIOL  OGY. 

definite  chromogenic  properties,  producing  various 
shades  of  yellow-  and  orange-colored  pigment,  while 
others  grow  with  a  pink  color. 

The  occurrence  of  bacterium  pseudodiphtheriticum  in 
pure  culture  in  superficial  abrasions  showing  a  slight 
tendency  to  suppuration  ;  the  fact  that  these  organisms, 
when  injected  into  the  peritoneal  cavity  of  guinea-pigs, 
produce  purulent  peritonitis;  that  such  organisms  are 
frequently  encountered  in  vaccine  virus  and  in  the  pus 
of  vaccination  wounds ;  and  that  frequently  in  cases  of 
mastitis  in  cows  such  organisms  occur  in  large  numbers 
in  pure  culture,  has  led  to  the  supposition  that  this 
group  of  organisms  was  probably  responsible  for  sup- 
purations occurring  under  certain  special  conditions. 
With  these  facts  in  mind  specimens  of  pus  were  derived 
from  thirty  cases  with  suppurating  wounds  in  the  Uni- 
versity of  Pennsylvania  Hospital,  and  careful  bacterio- 
logical examination  of  these  specimens  showed  the  pres- 
ence of  bacterium  pseudodipthheriticum  in  43  per  cent, 
of  the  cases.  These  organisms  were  always  found  in 
conjunction  with  one  or  more  of  the  group  of  pyogenic 
organisms,  and  it  is  impossible  to  state  how  much  of  the 
effect  was  due  to  any  one  of  the  organisms  present.  It 
seems  probable,  however,  in  the  light  of  what  has  been 
said,  that  these  bacteria  were  present  not  merely  as 
accidental  invaders,  but  that  in  some  way  they  contrib- 
uted toward  the  results. 

The  fact  that  some  of  the  organisms  isolated  from 
the  pus,  when  inoculated  into  the  peritoneal  cavity  of 
guinea-pigs,  show  distinct  pyogenic  properties  gives 
strong  support  to  the  opinion  that  this  group  is  of  greater 
importance  than  was  heretofore  supposed.  Repeated 
passage  through  guinea-pigs  serves  to  so  increase  the 


BACTERIUM  XEROSIS.  417 

pathogenic  properties  of  these  organisms  that  they  cause 
the  death  of  the  animal  in  less  than  twenty-four  hours 
with  marked  inflammatory  reaction  affecting  the  perito- 
neum as  well  as  the  abdominal  organs. 

The  morphologic  and  biologic  characters  of  some 
members  of  the  group  of  bacterium  pseudodiphtheriticum 
are  very  closely  allied  to  those  of  bacterium  diphtherias 
Other  members  of  the  group,  however,  are  readily  dif- 
ferentiated from  bacterium  diphtheria?  by  either  the 
morphologic  or  the  biologic  characters,  or  by  both.  Many 
of  the  members  of  the  group  produce  very  little  acid 
when  grown  in  carbohydrate  media,  and  the  slight 
degree  of  acidity  produced  is  frequently  obliterated  by 
a  marked  degree  of  alkali  production.  This  fact  is  of 
special  value  in  the  differentiation  from  bacterium  diph- 
theria'. 

BACTERIUM   XEROSIS  (NEISSER  AND   KUSCHBERT), 
MIGULA,  1900. 

Synonym  :  Bacillus  xerosis,  Neisser  and  Kuschbert,  1883. 

Another  organism  which  is  also  related  in  its  mor- 
phologic and  biologic  characters  to  bacterium  diphtheria) 
is  bacterium  xerosis,  first  encountered  by  Kuschbert  and 
Neisser  in  xerosis  of  the  conjunctiva,  and  which  has 
since  been  found  on  the  conjunctiva  by  a  number  of 
investigators,  in  various  diseases  as  well  as  in  health. 

The  xerosis  bacteria  are  less  likely  to  be  mistaken  for 
bacterium  diphtheria?  because  they  are  somewhat  smaller 
and  have  less  tendency  to  show  multiple  striations. 
Usually  they  stain  deeply  at  the  poles  with  only  one 
clear  unstained  band  in  the  centre.  It  is  only  occa- 
sionally that  a  few  striated  organisms  are  encountered 
in  a  culture. 

27 


418  BACTERIOLOGY. 

Biologically  bacterium  xerosis  is  readily  differentiated 
from  bacterium  diphtherias  because  of  the  scant  growth 
that  takes  place  on  the  ordinary  culture-media.  On 
agar-agar  the  growth  appears  as  small  transparent  colo- 
nies which  have  little  tendency  to  coalesce.  On  gelatin 
the  growth  is  slow,  and  frequently  shows  as  minute,  iso- 
lated colonies  along  the  needle  track.  In  litmus-milk  a 
slight  degree  of  acidity  is  produced.  In  bouillon  the 
growth  is  so  slight  as  to  leave  the  medium  practically 
unaltered.  The  growth  on  potato  is  slight  and  invisible. 

Differentiation  of  Members  of  the  Group. — Knapp  l 
reports  that  the  serum- water  media  of  Hiss,  to  which 
different  carbohydrates  have  been  added,  serve  to  differ- 
entiate between  bacterium  diphtheria,  bacterium  pseudo- 
diphtheriticum, and  bacterium  xerosis.  Bacterium  dipli- 
therice  ferments  dextrose,  mannite,  maltose,  and  dextrin 
with  formation  of  acid  and  the  coagulation  of  the  medium. 
Saccharose  is  not  fermented.  Bacterium  xerosis  fer- 
ments dextrose,  mannite,  maltose,  and  saccharose,  with 
formation  of  acid  and  coagulation  of  the  medium.  Dex- 
trin is  not  fermented.  Bacterium  pseudodiphtheriticum 
does  not  ferment  any  of  these  carbohydrates.  Knapp 
claims  that  a  positive  differentiation  of  the  organisms 
may  be  made  by  merely  inoculating  the  Hiss  media 
containing  dextrin  and  saccharose.  If  the  dextrin  is 
alone  fermented,  the  organism  is  bacterium  diphtheria?,  if 
only  the  saccharose  is  fermented,  the  organism  is  bacte- 
rium xerosisj  and  if  neither  of  these  carbohydrates  is  fer- 
mented, the  organism  is  bacterium  pseudodiphtheriticum. 

Through  the   suggestion  of  Neisser2  we  are  fortti- 

1  Knapp  :  Jour.  Med.  Research,  vol.  xii.,  p.  475,  1904. 

2  Neisser :  Zeitschrft  fur  Hygiene  und  Infektionskrankbeiten,  1897, 
Bd.  xxiv. 


BACTERIUM  XEROSIS.  419 

nately  enabled  to  differentiate  between  bacterium  diph- 
theria and  the  "  pseudo "  forms.  He  has  found  that 
by  the  use  of  a  particular  staining  method  the 
appearance  of  bacterium  diphtherice  is  strikingly 
unlike  that  of  the  confusing  forms.  His  differential 
method  comprehends  the  following  manipulations : 
the  culture  to  be  tested  should  be  grown  upon  Loffler's 
blood-serum  mixture  solidified  at  100°  C. ;  it  should 
develop  at  a  temperature  not  lower  than  34°  C.  and  not 
higher  than  36°  C. ;  and  it  should  be  not  younger  than  nine 
and  not  older  than  twenty-four  hours.  A  cover-glass  prep- 
aration made  from  such  a  culture  is  stained  as  follows : 

a.  It  is  subjected  to  the  following  mixture  for  from 
one  to  three  seconds : 

Methylene-blue  (Griibler's) 1  gramme. 

Alcohol  (96  per  cent.) ,.,...    20  c.c. 

When  dissolved,  mix  with 

Acetic  acid 50  c.c. 

Distilled  water 950  c.c. 

b.  After  thoroughly  rinsing  in  water,  it  is  stained  for 
from  three  to  five  seconds  in  vesuvin  (Bismarck-brown), 
2  grammes,  dissolved  in  1  litre  of  boiling  distilled  water, 
filtered,  and  allowed  to  cool.    It  is  again  rinsed  in  water 
and  examined  as  a  water-mount,  or  it  may  be  dried  and 
mounted  in  balsam. 

When  so  treated  the  diphtheria  bacterium  appears  as 
faintly  stained  brown  rods,  in  which  from  one  to  three 
dark-blue  granules  are  always  to  be  observed.  The 
dark  granules  are  at  one  or  both  poles  of  the  cell,  are 
more  or  less  oval,  and  usually  seem  to  bulge  a  little 
beyond  the  contour  of  the  bacterium  in  which  they  are 


420 


BACTERIOLOGY. 


located.  (See  Fig.  69.)  From  Neisser's  observations 
and  those  of  others,1  as  well  as  from  personal  experience, 
it  seems  safe  in  the  vast  majority  of  cases  to  regard  all 
bacteria  that  do  not  stain  in  the  way  described  as  distinct 
from  bacterium  diphtherice.2 

Blumenthal  and  Lipskerow3  compared  all  the  known 
staining  methods  that  had  been  suggested  for  the  differ- 
entiation between  bacterium  diphtherias  and  bacterium 
pseudodiphtheriticum.  They  decide  that  the  method 

FIG.  69. 


Bacterium  diphtherix,  stained  by  Neisaur's  method. 

which  yields  the  most  satisfactory  results  is  one  which 
had  not  heretofore  been  published,  but  which  Dr.  Lju- 
binsky  communicated  to  them.  This  method  consists  in 
the  fixation  of  the  preparation  for  from  one-half  to  two 

1  Frankel :  Berliner  klin.  Wochenschrift,  1897.  No.  50. 

2  Bergey :  Publications  of  the  University  of  Pennsylvania,  New  Series, 
No.  4, 1898. 

3  Blumenthal  and    Lipskerow :    Centralblatt   f.  Bacteriologie,    Bd. 
xxxviii.,  p.  359. 


BACTERIUM  XEROSIS.  421 

minutes  in  the  following  solutions  :  Pyoktanin  (Merck) 
0.25  grammes,  acid  acetic  (5  per  cent.)  100  c.c.  Washing 
in  water  and  counterstaining  with  a  1  to  1000  solution 
of  vesuvin  for  one-half  minute.  By  this  method  the 
polar  granules  of  bacterium  diphtheria?  are  stained  bluish 
black,  are  large,  and  may  be  seen  in  almost  all  of  the 
organisms.  The  contour  of  the  darkly  violet  stained 
bacterium  diphtherias  is  sharply  defined,  and  it  is  very 
easily  differentiated  from  any  other  organisms  that  may 
be  present  in  the  preparation. 

NOTE. — Prepare  cover-slip  preparations  from  the 
mouth-cavities  of  healthy  individuals  and  from  those 
having  decayed  teeth.  Do  they  correspond  in  any  way 
with  those  made  from  diphtheria  ?  Do  the  same  with 
different  forms  of  sore-throat.  Do  the  peculiarities  of 
any  of  the  organisms  suggest  those  of  bacterium  diph- 
theria?? Wherein  is  the  difference? 

In  cultures  and  cover-slips  made  from  both  diph- 
theritic and  from  innocent  sore-throats  are  any  organ- 
isms almost  constantly  present?  Which  are  they,  and 
what  are  their  characteristics  ? 

Which  are  the  predominating  organisms  in  the  an- 
ginas of  scarlet  fever? 

Do  these  organisms  simulate,  in  their  cultural  and 
morphological  peculiarities,  any  of  the  different  species 
with  which  you  have  been  working  ? 

Do  the  diphtheria  organisms  disappear  from  the  throat 
with  the  disappearance  of  the  membrane  ?  How  long 
do  they  persist?  When  obtained  from  the  throats  of 
convalescents  are  they  still  pathogenic  for  guinea-pigs  ? 

Prepare  a  bouillon  culture  of  virulent  bacillus  diph- 
theria? ;  after  it  lias  been  growing  for  thirty-six  hours 


422  S  A  CTERIOLOG  Y. 

at  37°-30°  C.  inoculate  a  guinea-pig  subcutaneously 
with  about  0.1  c.c.  of  it.  If  the  animal  dies,  note  care- 
fully the  findings  at  autopsy,  especially  the  distribution 
of  the  bacilli.  Now  add  to  this  culture  sufficient  pure 
carbolic  acid  or  trikresol  to  kill  all  bacteria  in  it,  and 
inject  under  the  skin  of  another  guinea-pig  varying 
amounts  of  the  culture  so  treated,  beginning  with  0.05 
c.c. ;  determine  the  minimum  fatal  dose,  and  note  in 
which  respects  the  post-mortem  findings  simulate  and 
in  which  they  differ  from  those  of  the  first  animal. 
Should  any  of  the  animals  survive  the  injections  of  the 
disinfected  culture,  note  carefully  their  condition  from 
day  to  day,  particularly  any  fluctuations  in  weight. 
When  they  have  quite  recovered  inoculate  them  with 
living,  virulent  diphtheria  organisms.  Do  the  results 
correspond  with  those  obtained  with  guinea-pigs  that 
have  never  been  treated  at  all  ?  Explain  the  results. 

DIPHTHERIA  ANTITOXIN. — As  stated  above,  the 
growth  of  bacterium  diphtheria?  is  accompanied  by  the 
elaboration,  of  a  poison  of  remarkable  toxicity  that  is 
accountable  for  the  constitutional  symptoms  and  patho- 
logical lesions  by  which  the  disease  is  characterized.  If 
by  appropriate  methods  this  poison  (toxin)  be  separated 
from  the  bacteria  by  which  it  was  formed,  it  is  capable, 
when  injected  into  susceptible  animals,  of  causing  death 
and  practically  all  the  lesions  that  accompany  the  dis- 
ease when  due  to  the  invasion  of  the  living  bacteria.  If, 
on  the  contrary,  the  dose  of  poison  be  so  adjusted  as  to 
cause  only  temporary  inconvenience  and  not  endanger 
life,  and  this  dose  be  injected  repeatedly,  gradually  in- 
creasing in  size  as  the  animal  is  able  to  bear  it,  after  a 
while  a  marked  tolerance  is  established,  so  that  the  animal 


DIPHTHERIA   ANTITOXIN.  423 

may  be  given  many  times  the  amount  of  the  toxin  that 
would  otherwise  prove  fatal — /.  <'.,  many  times  the  lethal 
dose  for  an  animal  that  had  not  acquired  such  a  tolerance. 

If  blood  be  now  drawn  from  the  animal  that  has 
become  habituated,  so  to  speak,  to  the  diphtheria  toxin, 
and  the  serum  collected  from  it,  we  discover  several 
important  facts,  viz.  : 

That  this  serum  when  mixed  with  the  previously 
determined  lethal  dose  of  the  toxin  in  a  test-tube  will 
either  neutralize  its  toxicity  or  greatly  reduce  it,  accord- 
ing to  the  amount  of  serum  used. 

That  if  we  inject  into  an  animal  the  determined  fatal 
dose  of  the  toxin,  and  immediately  afterward  inject  a 
quantity  of  the  serum,  either  the  animal  will  not  die  or 
the  death  will  be  more  or  less  delayed,  according  to  the 
amount  of  serum  employed. 

That  if  a  susceptible  animal  be  inoculated  with  a 
living  culture  of  virulent  bacterium  diphtheria?,  its  life 
may  be  saved,  or  its  death  postponed,  by  the  subsequent 
injection  of  the  serum ;  the  result  depending  upon  the 
amount  of  serum  used  and  the  lapse  of  time  between 
inoculation  with  the  bacteria  and  injection  of  the  serum. 

And,  finally,  that  although  this  serum  has  such  a 
marked  effect  upon  the  toxins  of  bacterium  diphtheria?  in 
a  test-tube  or  in  the  animal,  and  so  striking  an  influence 
upon  the  course  of  infection  with  the  living  organisms 
in  the  animal,  it  has  little  or  no  effect  upon  the  living 
bacteria  either  in  a  test-tube  or  at  the  site  of  inoculation 
in  the  living  animal  body. 

This  serum  with  which  we  have  been  experimenting 
is  the  so-called  "  diphtheria  antitoxin"  or  "  antidiph- 
theritie  serum." 

For  practical    purposes,  it  is  obtained  from   horses, 


424  BACTERIOLOGY. 

the  animals  being  treated  with  gradually  increasing 
doses  of  diphtheria  toxin  until  they  are  able  to  with- 
stand enormous  multiples  of  the  ordinarily  fatal  dose. 
When  this  point  is  reached,  the  protective  body — the 
antitoxin — is  present  in  the  blood  in  such  large  quan- 
tities that  the  serum  may  be  successfully  employed  in 
the  treatment  of  diphtheria  in  human  beings — /.  e.,  as 
an  antidote  to  the  diphtheria  toxin  that  is  produced  by 
the  growing  bacteria  in  the  throat,  or  else  where,  and 
distributed  through  the  body  by  the  circulating  blood. 

THE  STANDARDIZATION  OF  DIPHTHERIA  ANTI- 
TOXIN.— The  value  of  diphtheria  antitoxin  may  be  de- 
termined according  to  several  different  standards.  Those 
that  are  best  known  have  been  proposed  by  Behring  and 
Ehrlich. 

1.  Behrincfs  Method. — He  designates  as  a  "normal" 
poison  a  toxin  of  which  0.01  c.c.  suffices  to  kill  a  guinea- 
pig  weighing  250  grammes  in  four  days.  Of  such  a 
normal  diphtheria  toxin  1  c.c.  will  be  sufficient  to  kill 
100  guinea-pigs  weighing  250  grammes  each,  or  25,000 
grammes  in  weight  of  guinea-pigs. 

The  quantity  of  antitoxin  that  is  required  to  just  pro- 
tect 25,000  grammes  weight  of  guinea-pigs  from  the 
minimum  fatal  dose  of  the  toxin  is  called  one  immuni- 
zing unit.  If  an  immune  serum  contains  in  1  c.c.  one 
immunizing  unit,  it  represents  a  "  normal "  antitoxin. 

To  determine  the  strength  of  an  immune  serum,  1  c.c. 
of  normal  toxin  is  mixed  with  increasing  quantities  of 
the  serum,  and  these  mixtures  are  injected  subcutaneously 
into  guinea-pigs ;  the  quantity  of  the  serum  which  suf- 
fices to  neutralize  that  amount  of  normal  toxin — i.  e., 
that  keeps  the  animal  alive  for  four  days  or  longer — 
contains  one  immunizing  unit. 


DIPHTHERIA   ANTITOXIN.  425 

2.  Ehrlich's  Method. — Ehrlich  has  recently  introduced 
the  use  of  a  standard  diphtheria  antitoxin  in  a  dry  state 
which  contains  1700  immunizing  units  in  each  gramme. 
This  standard  antitoxin  is  distributed  by  the  Institute 
for  testing  serum  at  Frankfort-on-the-Main,  and  is  now 
being  used  in  a  great  many  places  for  the  standardiza- 
tion of  diphtheria  antitoxin.  A  test  toxin  is  prepared, 
corresponding  to  this  standard  antitoxin,  and  with  this 
toxin  the  strength  of  the  unknown  serum  is  titrated. 

If,  for  instance,  the  test  toxin  is  of  such  a  strength 
that  0.003  c.c.  represents  the  minimum  fatal  dose  for  a 
guinea-pig  of  250  grammes,  then  0.3  c.c.  would  represent 
100  times  the  minimum  fatal  dose  of  toxin,  and,  accord- 
ing to  Ehrlich's  standard,  an  immunity  unit  is  that 
amount  of  antitoxic  serum  which  will  neutralize  100 
times  the  minimum  fatal  dose  of  toxin.  In  performing 
the  test  to  estimate  the  strength  of  an  antitoxic  serum, 
the  antitoxin  is  diluted  with  sterile  water  in  varying 
proportions,  and  a  series  of  guinea-pigs  are  injected  with 
mixtures  of  100  times  the  minimum  fatal  dose  of  the 
toxin  and  varying  quantities  of  the  diluted  antitoxic 
serum.  For  this  purpose  guinea-pigs  of  approximately 
250  grammes  weight  are  employed.  If,  for  instance,  a 
guinea-pig  receiving  100  times  the  minimum  fatal  dose 
of  toxin,  and  0.1  c.c.  of  the  diluted  antitoxic  serum,  sur- 
vives for  four  days,  then  0.1  c.c.  of  the  serum  represents 
an  immunity  unit  of  antitoxin. 

An  antitoxic  serum  of  this  strength,  therefore,  contains 
10  times  the  normal  amount  of  antitoxin,  because  it  con- 
tains the  immunity  unit  in  only  0.1  c.c. ;  a  normal  anti- 
toxin being  one  in  which  an  immunity  unit  is  contained 
in  one  cubic  centimetre.  Antitoxic  serums  are  frequently 
of  such  high  degree  of  potency  that  they  contain  from 
800  to  1000  immunity  units  in  each  cubic  centimetre. 


CHAPTER    XX. 

Typhoid  fever — Study  of  the  organism  concerned  in  its  production — 
Its  morphological,  cultural,  and  pathogenic  properties — Bacillus 
coli — Bacillus  paratyphosus — Its  resemblance  to  Bacillus  typhoms. 

BACILLUS   TYPHOSUS. 

THE  organism  discovered  in  the  tissues  of  typhoid 
cadavers  microscopically  by  Eberth  (1880-81),  and 
subsequently  isolated  in  pure  culture  and  described  by 
Gaffky  (1884),  is  now  generally  recognized  as  the  etio- 

FIG.  70.  FIG.  71. 


M  >      %V 

.  \t*>>* 

s-.-l 


Bacillus  typhosus,  from  culture  Bacillus  typhows,  showing  flagella 

twenty-four  hours  old,  on  agar-  stained  by  Loffler's  method, 

agar. 

logical  factor  in  the  production  of  typhoid  fever.     It 
may  be  described  as  follows  : 

It  is  a  bacillus  about  three  times  as  long  as  broad, 
with  rounded  ends.  It  may  appear  at  one  time  as  very 
short  ovals,  at  another  time  as  long  threads,  and  both 

426 


BACILLUS  TYPHOSUS.  427 

forms  may  occur  together.  Its  breadth  remains  toler- 
ably constant.  Its  morphology  presents  little  that 
will  aid  in  its  identification.  (See  Fig.  70.)  It  stains  a 
trifle  less  readily  with  the  aniline  dyes  than  do  most  of 
the  other  organisms.  It  is  very  actively  motile,  and 
when  stained  by  the  special  method  of  Lbffler  (see 
this  method  in  chapter  on  Staining)  is  seen  to  possess 
very  delicate  locomotive  organs  in  the  form  of  fine, 
hair-like  flagella,  attached  in  large  numbers  to  all  parts 
of  its  surface.  (See  Fig.  71.)  These  flagella  are  not 
seen  in  unstained  preparations,  nor  are  they  rendered 
visible  by  ordinary  methods  of  staining. 

In  patients  suffering  from  typhoid  fever  the  organ- 
ism has  been  found  during  life  in  the  blood,  urine,  and 
faeces,  and  at  autopsies  in  the  tissues  of  the  spleen,  liver, 
kidneys,  intestinal  lymphatic  glands,  and  intestines. 

GELATIN  PLATES. — Its  growth,  when  seen  in  the 
depths  of  the  medium,  presents  nothing  characteristic, 
appearing  simply  as  round  or  oval,  finely  granular 
points.  On  the  surface  it  develops  as  very  superficial, 
blue- white  colonies,  with  irregular  borders.  They  are 
a  little  denser  at  the  centre  than  at  the  periphery. 

FIG.  72. 


Colony  of  bacillus  typhosus  on  gelatin. 


When  magnified,  the  colonies  present  wrinkles  or  folds, 
which  give  to  them,  in  miniature,  the  appearance  seen 
in  the  relief  maps  made  to  represent  mountainous  dis- 


428  BACTERIOLOGY. 

tricts.  (Fig.  72.)  These  colonies  have  sometimes  the 
appearance  of  flattened  pellicles  of  glass-wool,  and 
usually  a  pearl-like  lustre. 

On  AGAR-AGAR  the  colonies  present  nothing  typical. 

STAB-CULTURES.  —  In  stab-cultures  the  growth  is 
mostly  on  the  surface,  there  being  only  a  very  limited 
development  down  the  track  made  by  the  needle.  The 
surface-growth  has  the  same  appearance  in  general  as 
that  given  for  the  colonies. 

POTATO. — The  growth  on  potato  is  usually  described 
as  luxuriant  but  invisible,  making  its  presence  evident 
only  by  the  production  of  a  slight  increase  of  moisture 
at  the  inoculated  point,  and  by  a  limited  resistance 
offered  to  a  needle  when  it  is  scraped  across  the  track 
of  growth.  While  this  is  so  in  many  cases,  yet  it  cannot 
be  considered  as  invariable,  for  at  times  this  organism 
develops  more  or  less  visibly  on  potato. 

POTATO-GELATIN. — The  growth  is  similar  to  that 
upon  ordinary  nutrient  gelatin. 

MILK. — It  does  not  cause  coagulation  when  grown 
in  sterilized  milk. 

BOUILLON. — It  causes  uniform  clouding  of  the  bouil- 
lon and  brings  about  a  slightly  acid  reaction. 

INDOL  FORMATION. — It  is  customary  to  regard  this 
organism  as  devoid  of  the  power  of  forming  indol ; 
in  fact,  this  has  hitherto  been  considered  one  of  its 
important  differential  peculiarities.  By  the  usual  meth- 
ods of  cultivation  and  testing  the  indol  reaction  is  not 
observed  in  cultures  of  the  typhoid  bacillus.  It  has 
recently  been  shown,  however,  by  Peckham,  that  by 
repeated  transplantation,  at  short  intervals,  into  either 
Dunham's  peptone  solution,  or,  preferably,  a  freshly 
prepared  alkali-tryptone  solution,  made  from  trypton- 


BACILLUS  TYPHOSUS.  429 

ized  beef-muscle,  that  the  indol-producing  function 
may  be  induced  in  the  genuine  typhoid  bacillus  obtained 
directly  from  the  spleens  of  typhoid  cadavers.1 

It  does  not  produce  gaseous  fermentation.  On  lactose- 
litmus-agar-agar  it  grows  as  pale-blue  colonies,  causing 
no  reddening  of  the  surrounding  medium ;  though  if 
glucose  be  substituted  for  lactose,  both  the  colonies  and 
the  surrounding  medium  may  become  red.  In  the  fer- 
mentation-tube, in  glucose  or  lactose  bouillon,  no  evo- 
lution of  gas  as  a  result  of  fermentation  occurs. 

It  does  not  form  spores.  The  irregularities  of  stain- 
ing so  commonly  seen  in  this  organism  have  in  some 
instances  led  to  the  belief  that  the  pale,  unstained  por- 
tions of  the  bacilli  indicate  the  presence  of  spores. 
More  reliable  tests,  however,  have  demonstrated  the 
error  of  this  opinion.  (What  is  the  most  trustworthy 
test  of  spore-formation  ?) 

It  grows  at  any  temperature  between  20°  and  38°  C., 
though  more  favorably  at  the  latter  point.  It  is 
very  sensitive  to  high  temperatures,  being  killed  by 
an  exposure  of  ten  minutes  to  60°  C.,  and  in  a  much 
shorter  time  to  slightly  higher  temperatures. 

It  does  not  liquefy  gelatin. 

It  grows  both  with  and  without  oxygen. 

It  does  not  grow  rapidly. 

Owing  to  a  tendency  to  retraction  of  its  protoplasm 
from  the  cell-envelope  and  the  consequent  production 
of  vacuoles  in  the  bacilli,  the  staining  of  this  organism 
is  frequently  more  or  less  irregular.  At  some  points  in 
a  single  cell  marked  differences  in  the  intensity  of  the 

1  A.  W.  Peckham :  "The  Influence  of  Environment  Upon  the 
Biological  Functions  of  the  Colon  Group  of  Bacilli,"  Journal  of  Experi- 
mental Medicine,  1897,  vol.  ii. 


430  BACTERIOLOGY. 

staining  will  be  seen,  and  here  and  there  areas  quite 
free  from  color  can  commonly  be  detected.  These 
colorless  portions  are  often  so  sharply  defined  that  they 


FIG. 73. 


Diagrammatic  representation  of  retraction  of  protoplasm,  with  production 
of  pale  points,  in  bacillus  typhosus. 

look  as  if  they  had  been  punched  out  with  a  sharp 
instrument.  (See  Fig.  73.) 

PRESENCE  IN  TISSUES.— It  is  not  easy  to  demonstrate 
this  organism  in  tissues  unless  it  is  present  in  large  num- 
bers. The  manipulations  to  which  the  sections  are  sub- 
jected •  in  being  mounted  oftai  rob  the  bacilli  of  their 
stain,  and  render  them  invisible,  or  nearly  so.  If, 
however,  sections  be  stained  in  the  carbol-fuchsin  solu- 
tion, either  at  the  ordinary  temperature  of  the  room  or 
at  a  higher  temperature  (40°  to  45°  C.),  then  washed 
in  absolute  alcohol,  and  cleared  in  xylol  and  mounted 
in  balsam,  the  bacilli  (particularly  if  the  tissue  be  the 
liver  and  spleen)  can  readily  be  detected,  massed  to- 
gether in  their  characteristic  clumps.  If  used  in  the 
same  way,  the  alkaline  methylene-blue  solution  gives 
also  very  satisfactory  results. 

In  searching  for  the  typhoid  bacilli  in  tissues  this 
peculiar  deposition  in  clumps  must  always  be  borne  in 
mind,  otherwise  much  labor  will  be  expended  in  vain. 
In  tissues  the  typhoid  bacilli  do  not  lie  scattered  about 
in  the  same  way  as  do  the  organisms  in  tissues  from 
certain  other  conditions — septicaemia,  for  instance ;  they 


BACILLUS  TYPHOSUS.  431 

are  not  of  necessity  distributed  along  the  course  of  the 
capillaries,  but  are  localized  in  small  clumps  through 
the  organs,  and  it  is  for  these  clumps,  which  are  easily 
detected  under  a  low-power  objective,  that  one  should 
search.  This  peculiar  clumping  of  the  typhoid  bacilli 
in  the  tissues  cannot  be  satisfactorily  explained.  It 
may  possibly  be  due  to  the  specific  clumping  or  agglu- 
tinating influence  that  typhoid  blood  has  been  shown  to 
have  upon  the  typhoid  bacillus,  a  phenomenon  that  is 
readily  demonstrable  in  the  test-tube  or  under  the 
microscope.  In  other  words,  may  it  not  be  simply  the 
result  of  an  intracapillary  "  Widal  reaction "  ?  (See 
Widal  Reaction.) . 

When  the  section  is  prepared  for  examination,  if  it 
be  gone  over  with  a  low-power  objective,  one  will 
notice  at  irregular  intervals  little  masses  that  look  in 
every  respect  like  particles  of  stain  ing-matter  which 
have  been  precipitated  upon  the  section  at  that  point. 
When  these  masses  are  examined  with  a  higher  power 
objective  they  will  be  found  to  consist  of  small  ovals  or 
short  rods  so  closely  packed  that  the  individuals  com- 
posing the  clump  can  often  be  seen  only  at  the  extreme 
periphery  of  the  mass.  This  is  the  characteristic  ap- 
pearance of  the  typhoid  organism  in  tissues.  The  little 
masses  are  usually  in  the  neighborhood  of  a  capillary. 

RESULT  OF  INOCULATION  INTO  LOWER  ANIMALS. — 
A  great  many  experiments  have  been  made  in  a  variety 
of  ways  with  the  view  of  reproducing  the  pathological 
conditions  of  this  disease,  as  seen  in  man,  in  the  tis- 
sues of  lower  animals,  but  with  practically  no  success. 
From  the  time  of  its  discovery  up  to  within  a  compara- 
tively recent  date  there  was  an  almost  continuous  con- 
troversy concerning  the  infective  properties  of  bacillus 


•432  BACTERIOLOG  Y. 

typhoxus  for  animals.  By  some  it  was  held  that  the 
effects  of  its  introduction  into  animals  were  manifestly 
of  toxic l  origin,  while  others  regarded  them  as  evidences 
of  genuine  infection.2  These  diversities  of  opinion  are 
hardly  surprising  when  we  remember  that  animals 
never  suffer  naturally  from  a  disease  similar  to  typhoid 
fever,  and  therefore  offer  many  obstacles  to  its  faithful 
reproduction,  and  that  the  vigor  of  this  organism  when 
cultivated  from  various  sources  is  liable  to  a  wide  range 
of  fluctuation.  For  a  time  there  seemed  to  be  good 
grounds  for  the  opinion  that  under  exceptional  circum- 
stances bacillus  ti/phosm  did  exhibit  truly  infective 
properties,  and  the  reported  experiments  of  Cygnseus3 
in  particular,  as  well  as  a  single  observation  by  the 
writer,4  in  no  wise  weakened  this  opinion.  By  a  variety 
of  methods  CygnaBiis  demonstrated  that  this  organism 
possessed  the  property  of  multiplying  within  the  in^ 
ternal  organs  of  animals  and  of  causing  constitutional 
symptoms  and  pathological  lesions  that  very  closely 
simulated  those  of  typhoid  fever  as  seen  in  man.  In 
1890  the  writer  called  attention  to  the  lesions  found  in 
one  of  a  number  of  rabbits  that  had  succumbed  to 
intravenous  injection  of  large  amounts  of  fluid  cultures 
of  this  organism. 

In  this  case  there  was  an  ulcer  in  the  ileum  which 
was  macroscopically  and  microscopically  identical  with 
those  found  at  autopsy  in  the  small  intestine  of  human 

1  Toxic — poisonous  results  not  necessarily  accompanied  by  the  growth 
of  organisms  throughout  the  tissues. 

2  Infective  or  septic — poisoning  of  the  tissues  as  a  result  of  the  growth 
of  bacteria  within  them. 

3Cygnfeus:  Ziegler's  Beitrage  zur  Anat.  und   Path.,  1890,  Bd.  vii. 
Heft  3,  S.  377. 
4  Bulletin  of  the  Johns  Hopkins  Hospital,  1890,  vol.  i.  p.  63. 


BACILLUS  TYPHUS  US.  433 

subjects  dead  of  this  disease.  The  typhoid  bacilli  were 
not  only  obtained  from  the  spleen  of  the  animal  by 
culture  method,  but  the  characteristic  clumps  were  also 
demonstrated  microscopically  in  sections  of  the  organ. 

It  must  be  said,  however,  that  such  results  are  ex- 
tremely rare.  As  a  rule,  the  only  effects  that  follow 
the  introduction  of  this  organism  into  animals  are  refer- 
able to  the  intoxicating  action  of  the  materials  used. 
In  fact,  the  results  of  modern  investigations  have  placed 
bacillus  typhosus  in  the  category  of  toxin-producers, 
and  through  the  use  of  the  toxins  produced  by  it  ani- 
mals have  been  rendered  immune  from  otherwise  fatal 
doses.  The  serum  of  such  animals  has  also  been  shown 
to  possess  a  certain  degree  of  immunizing  power.1 

In  connection  with  the  inoculation  of  animals  with 
bacillus  typhosus  observations  of  a  most  important 
nature  have  been  made  by  Sanarelli 2  upon  the  arti- 
ficial induction  of  susceptibility  to  its  pathogenic  ac- 
tion. He  found  that  rabbits,  guinea-pigs,  and  mice 
could  be  rendered  susceptible  to  infection  by  this  organ- 
ism by  preliminary  injections  into  them  of  the  products 
of  growth  of  certain  saprophytes— bacillus  vulgaris, 
bacillus  prodigiosus,  and  bacillus  coli;  and  that  by 
whatever  means  the  animal  was  subsequently  inocu- 
lated with  fresh  cultures  of  the  typhoid  bacillus,  either 
into  the  circulation  or  into  the  peritoneal  cavity,  death 
resulted  in  from  twelve  to  forty-eight  hours,  with 
the  pathological  alterations  most  conspicuous  in  the 
digestive  tract,  and  particularly  in  the  small  intestine. 
In  these  cases  the  infection  is  general,  and  the  organisms 

1  Pfeiffer  and  Kolle :  Zeitschrift  fur  Hygiene  und  Infektionskrank- 
heiten,  1896,  Bd.  xxi.  S.  208. 

2  Sanarelli:  Annales  de  1'Institut  Pasteur,  1892,  tome  vi. 

28 


434  BACTER10LOG  Y. 

may  be  recovered  from  the  blood  and  internal  organs. 
It  is  the  opinion  of  Sanarelli  that  the  toxic  conditions 
produced  by  the  preliminary  injections  of  the  products 
of  growth  of  the  saprophytic  organisms  may  be  consid- 
ered analogous  to  a  similar  condition  that  may  occur 
in  man  from  the  absorption  of  abnormal  products  of 
fermentation  from  the  intestinal  canal — an  auto-intoxi- 
cation that  so  reduces  the  resistance  of  the  individual 
as  to  render  him  susceptible  to  infection  by  the  bacillus 
of  typhoid  fever,  should  it  gain  access  to  his  alimentary 
tract. 

Alessi1  reports  that  rats,  guinea-pigs,  and  rabbits, 
when  compelled  to  breathe  the  gaseous  products  of 
decomposition  from  the  contents  of  a  cesspool,  or  from 
other  decomposing  matters,  gradually  became  susceptible 
to  infection  by  the  typhoid  bacillus ;  but,  unfortunately 
for  the  value  of  this  observation,  the  description  given 
by  Alessi  of  the  two  cultures  of  so-called  typhoid  bacilli 
used  by  him  for  inoculation  was  in  one  case  certainly 
not  that  of  the  typhoid  organism,  and  in  the  other  the 
culture  used  had  been  kept  under  artificial  conditions 
so  long  as  hardly  to  be  reliable  for  tests  of  this 
character. 

The  importance  of  these  observations  in  their  bearing 
upon  the  etiology  of  typhoid  fever,  if  they  are  demon- 
strated by  subsequent  experiment  to  be  trustworthy,  is 
too  obvious  to  necessitate  emphasis,  and  it  is  greatly  to 
be  desired  that  they  may  not  be  permitted  to  pass  un- 
noticed, but  that  others  interested  may  find  occasion  to 
institute  experiments  in  the  same  direction,  with  the 
hope  that  some  light  may  be  shed  upon  the  mooted 

1  Alessi :    Centralblatt  fur  Bakteriologie  un.  Parasitenkunde  1894, 
Bd.  xv.  No.  7,  p.  228. 


BACILLUS  TYPHOSUS.  435 

question  concerning  tlie  influence  of  gaseous  products 
of  decomposition  upon  the  health  of  individuals,  and 
particularly  upon  the  part  played  by  them  in  diminish- 
ing natural  resistance  to  infection.1 

Because  of  the  variations  in  the  morphology  and  cult- 
ural peculiarities  of  this  organism,  and  because  of  the 
difficulty  experienced  in  efforts  to  reproduce  in  lower 
animals  the  conditions  found  in  the  human  subject, 
typhoid  fever  is  bacteriologically  one  of  the  most  unsat- 
isfactory of  the  infectious  diseases. 

A  number  of  other  organisms  appear  botanically  to 
be  closely  related  to  the  typhoid  bacillus,  and  with  our 
present  methods  for  studying  them  they  so  closely  simu- 
late it  that  the  difficulty  of  identifying  this  organism 
is  sometimes  very  great.  In  addition  the  variability 
constantly  seen  in  pure  cultures  of  the  typhoid  bacillus 
itself  in  no  way  renders  the  task  more  simple. 

For  example,  the  morphology  of  the  typhoid  ba- 
cillus is  conspicuously  inconstant ;  its  growth  on  potato, 
which  was  formerly  described  as  characteristic,  may, 
with  the  same  stock,  at  one  time  be  the  typical  invis- 
ible development,  at  another  time  it  may  grow  in  a  way 
easily  to  be  seen  with  the  naked  eye  ;  and  the  change  of 
reaction  which  it  is  said  to  produce  in  bouillon  is  some- 
times much  more  intense  than  at  others.  The  indol- 
producing  function,  hitherto  regarded  as  absent  from 
this  organism,  is  now  known  to  be  occasionally  de- 
monstrable by  ordinary  methods,  and  frequently  by 
special  methods  of  cultivation.  (Peckham,  I.  c.)  The 

1  See  paper  by  the  author :  "  The  Effects  of  the  Gaseous  Products  of 
Decomposition  upon  the  Health,  and  Resistance  to  Infection,  of  Certain 
Animals  that  are  Forced  to  Respire  Them,"  Transactions  of  the  Asso- 
ciation of  American  Physicians,  1895,  vol.  x.  pp.  16-44. 


436  BACTERIOLOGY. 

only  properties  possessed  by  it  that  may  be  said  to  be 
constant  are  its  motility ;  its  inability  to  cause  gaseous 
fermentation  of  glucose,  lactose,  or  saccharose ;  its  inca- 
pacity for  coagulating  milk;  and  its  growth  on  gelatin 
plates ;  but  there  are  other  bacilli  which  possess  these 
same  characteristics  to  a  degree  that  renders  their  differ- 
entiation from  the  typhoid  organism  often  a  matter  that 
requires  the  careful  application  of  all  the  different  tests. 

THE  AGGLUTINATION  REACTION. — An  interesting 
reaction  of  the  typhoid  bacillus  is  seen  when  it  is 
brought  in  contact  with  the  blood-serum  from  human 
beings  sick  of  typhoid  fever,  or  from  animals  that 
have  survived  inoculation  with  cultures  of  this  organ- 
ism. This  reaction  consists  of  a  peculiar  alteration 
in  the  relation  of  the  organisms  to  one  another  in 
the  fluid.  As  ordinarily  seen  in  a  hanging  drop  of 
bouillon,  the  typhoid  bacilli  appear  as  single,  act- 
ively motile  cells ;  when  to  such  a  drop  a  drop  of  di- 
lute serum  from  a  case  of  typhoid  fever  is  added  the 
motility  of  the  organism  gradually  becomes  lessened, 
and  finally  ceases,  and  the  bacteria  congregate  in 
larger  and  smaller  clumps.  The  reaction  may  also  be 
produced  in  another  way,  viz.,  by  adding  to  about  4  or 
5  c.c.  of  a  twenty-four-hour-old  bouillon  culture  of 
typhoid  bacilli  in  a  narrow  test-tube  about  eight  drops 
of  serum  from  a  case  of  typhoid  fever,  after  which  the 
tube  is  placed  in  the  incubator.  After  a  few  hours  the 
normally  clouded  culture  is  seen  to  have  undergone  a 
change ;  instead  of  the  diffuse  cloud  caused  by  the  growth, 
the  fluid  is  found  clear  and  contains  within  it  flocculent 
masses  of  the  bacteria  that  have  agglutinated  together 
as  a  result  of  the  specific  action  of  the  serum  used. 

For  the  hanging-drop  test,  sufficient  serum  may  be 
obtained  from  a  needle-prick  in  the  finger,  while  for 


BACILLUS  TYPHOSUS.  437 

the  test-tube  reaction  a  larger  amount  is  needed  ;  this 
may  be  obtained  from  blood  drawn  from  a  superficial 
vein  by  means  of  a  hypodermic  syringe,  or  from  the 
cleansed  skin  by  a  wet-cup,  or,  better  still,  from  a 
small  cantharides  blister. 

It  is  proper  to  state,  however,  that  occasionally  cult- 
ures of  genuine  typhoid  bacilli  are  encountered  that  do 
not  respond  to  this  peculiar  influence  of  typhoid  blood, 
even  though  the  blood  be  tested  at  different  stages  of 
the  disease,  and  even  though  it  causes  the  characteristic 
cessation  of  motion  and  clumping  with  other  cultures  of 
this  organism  upon  which  it  may  be  tried. 

When  employed  conversely — i.  e.,  for  deciding  if 
the  serum  used  is  from  a  case  of  typhoid  fever  or  not — 
the  reaction  constitutes  "WidaPs  serum  diagnosis  of 
typhoid  fever/'  For  this  purpose  it  is  often  necessary 
to  test  several  cultures  of  genuine  typhoid  bacilli,  from 
different  sources  and  of  varying  degrees  of  vitality,  be- 
fore a  culture  is  procured  that  gives  the  reaction  most 
conspicuously  and  quickly  with  genuine  typhoid  serum. 
This  culture  is  then  to  be  set  aside,  to  be  used  for  this 
test  with  serums  from  doubtful  cases  of  the  disease. 

WIDAI/S  REACTION  WITH  DRIED  BLOOD. — For  clin- 
ical purposes  it  is  of  importance  to  know  that  this  reac- 
tion can  be  obtained  from  dried  blood — i.  e.,  by  the 
method  suggested  by  Wyatt  Johnston,  of  Montreal. 
In  this  method  a  drop  of  the  blood  to  be  tested,  ob- 
tained by  a  needle-prick  in  the  cleansed  finger  or  lobe 
of  the  ear,  is  collected  on  a  bit  of  clean,  unglazed 
paper  and  allowed  to  dry.  The  paper  is  then  folded, 
kept  free  from  contamination,  and  taken  to  the  labora- 
tory. With  a  medium-size  platinum-wire  loop  a  drop 
of  sterile  bouillon,  \vater,  or  physiological  salt  solution 


438  BACTERIOLOG  Y. 

is  gently  rubbed  upon  the  drop  of  dried  blood  until  tin- 
contents  of  the  loop  are  of  a  dark  amber  color;  this  is 
then  mixed  with  a  drop  of  a  bouillon  culture  of  typhoid 
bacilli  on  a  cover-glass,  which  is  mounted  upon  the 
hollow-ground  slide  as  a  hanging  drop,  when  the  effect 
of  the  diluted  blood  upon  the  culture  can  be  observed 
with  the  microscope.  The  reaction,  if  positive,  should 
occur  within  a  half  hour.  Many  object  to  this  method 
because  it  is  impossible  accurately  to  dilute  the  blood 
by  the  plan  used.  A  number  of  tests  have  shown  us 
that  preparations  made  in  this  way  correspond  roughly 
with  a  fresh-blood  dilution  of  from  1  :  1  o  to  1  :  20,  as 
determined  by  the  haemoglobinometer.  In  a  small 
number  of  cases  in  which  parallel  tests  were  made 
with  this  and  with  fresh  fluid  serum  the  results  were 
concordant.  We  are  inclined  to  the  opinion,  however, 
that  in  doubtful  cases,  in  which  all  the  available  clin- 
ical evidence  is  opposed  to  either  the  positive  or  nega- 
tive results  of  the  test,  the  difficulty  is  much  more 
certainly  cleared  away  by  the  use  of  highly  diluted 
and  exactly  diluted  fresh  serum  than  by  this  method. 
Competent  observers  are  of  the  opinion  that  in  all 
such  cases  the  quantity  of  serum  in  the  hanging  drop 
should  be  decreased  until  it  is  present  in  the  proportion 
of  from  (not  less  than)  1  :  50  to  1  :  60,  and  that,  if  after 
exposure  to  this  dilution  for  two  hours  the  bacilli  are 
still  motile  and  not  clumped  together,  or  the  reaction  is 
deficient  in  only  one  or  the  other  of  these  peculiarities, 
the  case  from  which  the  serum  was  obtained  may  be  safely 
regarded  as  not  typhoid  fever,  or  if  typhoid  the  exami- 
nation was  not  made  at  a  time  when  agglutinin  was  pres- 
ent in  demonstrable  quantities  in  the  circulating  blood. 
Experience  with  the  dry-blood  method  at  the  Mu- 


BACILLUS  TYPHOSUS.  439 

nicipal  Laboratory  of  Philadelphia  in  more  than 
12,000  examinations  from  about  10,000  febrile  con- 
ditions, leads  us  to  regard  the  culture  used  as  one  of 
the  most  important  factors  in  the  test.  After  deciding 
upon  the  most  suitable  culture  for  the  reaction — and  it 
is  often  necessary  to  try  a  great  number  from  various 
sources — we  have  adopted  the  plan  of  daily  trans- 
planting the  culture  into  fresh  bouillon  and  keep- 
ing it  at  a  temperature  rarely  above  20°-22°  C.  The 
bacilli  grown  under  these  circumstances  are  usually 
somewhat  longer  than  when  cultivated  at  higher  tem- 
perature, and  they  exhibit  a  regular,  gliding  motility 
that  renders  it  more  easy  to  follow  the  individual  cells 
under  the  microscope  than  when  they  possess  the  usual 
active,  darting  motion. 

In  the  group  of  cases  examined  by  us  by  the  dry- 
blood  method,  including  typhoid  and  other  febrile  con- 
ditions, there  is  a  discrepancy  between  the  clinical  and 
the  laboratory  diagnosis  in  from  2  to  3  per  cent,  of  the 
cases  examined. 

In  the  hands  of  all  who  have  carefully  employed 
the  Widal  reaction  for  the  diagnosis  of  typhoid  fever 
the  results  are  reported  to  have  been  almost  uniformly 
satisfactory.  In  the  great  majority  of  cases  the  reac- 
tion is,  so  far  as  experience  indicates,  specific — i.  e.,  a 
typical  reaction  does  not  occur  between  typhoid  serum 
and  organisms  other  than  the  typhoid  bacillus,  nor  be- 
tween the  typhoid  bacillus  and  serums  other  than  those 
of  typhoid  fever.  There  are,  however,  confusing  reac- 
tions— so-called  pseudo-reactions — in  which  more  or 
less  clumping  of  the  bacilli  and  a  diminution  of  motion, 
without  complete  cessation,  are  observed.  These  reac- 
tions have  been  seen  to  occur  with  normal  blood  and 


440  BACTERIOLOGY. 

with  blood  from  other  febrile  conditions.  It  is  said  by 
Johnston  and  McTaggart1  that  they  can  be  prevented 
if  cultures  of  just  the  proper  degree  of  vitality  are  em- 
ployed ;  and  this  corresponds  with  the  results  of  a  fairly 
wide  personal  experience  with  the  test. 

The  blood  of  certain  animals,  as  well  as  a  number  of 
chemical  substances,  such  as  corrosive  sublimate,  alco- 
hol, salicylic  acid,  resorcin,  and  safranin  in  high  dilution, 
cause  agglutination  of  the  typhoid  bacilli ;  but  the  reac- 
tion is  not  specific,  for  in  most  cases  they  have  the  same 
effect  on  other  motile  bacilli. 

The  method  is  still  in  the  experimental  stage,  and 
there  are  numerous  features  not  entirely  clear.  In 
the  light  of  present  experience,  however,  it  is  fair 
presumptive  evidence  that  the  serum  is  from  a  case 
of  typhoid  fever  when  unmistakable  agglutination  and 
cessation  of  motion  are  seen  in  from  fifteen  to  twenty 
minutes  after  typhoid  bacilli  are  mixed  with  the  serum 
of  a  suspicious  febrile  condition. 

All  the  points  with  regard  to  morphologic  and  biologic 
characters  of  bacillus  typhosus,  and  of  the  organisms 
closely  resembling  it,  should  be  borne  in  mind  in  the 
examination  of  drinking-water  supposed  to  be  con- 
taminated by  typhoid  dejections,  for  the  organisms 
which  most  closely  approach  the  typhoid  bacillus  in 
growth  and  morphology  are  just  those  organisms  which 
would  appear  in  water  contaminated  from  cesspools — 
/.  e.j  the  organisms  constantly  found  in  the  normal  intes- 
tinal tract.  Even  in  the  stools  of  typhoid-fever  patients 
the  presence  of  these  normal  inhabitants  of  the  intes- 
tinal tract  renders  the  isolation  of  the  typhoid  organisms 
somewhat  troublesome. 

1  Johnston  and  McTaggart :  Montreal  Medical  Journal,  March,  1897. 


ISOLATING   THE  TYPHOID  BACILLUS.        441 

METHODS  OF  ISOLATING  THE  TYPHOID  BACILLUS. 
— Bacillus  iyphosus  is  so  variable  in  many  of  its  bio- 
logical peculiarities,  and  is  so  closely  simulated  in  cer- 
tain respects  by  a  group  of  other  organisms  to  which  it 
appears  to  be  botanically  related,  that  its  identification, 
especially  outside  the  infected  body,  is  usually  a  matter 
of  considerable  difficulty  and  uncertainty.  For  these 
reasons  many  efforts  have  been  made  to  discover  specific 
reactions  for  the  organism,  and  with  this  end  in  view 
many  methods  have  been  devised  for  its  isolation  from 
water,  faeces,  sewage,  and  other  matters  believed  to  con- 
tain it.  None  of  them,  however,  has  given  general  satis- 
faction, and  many  have  proved  wholly  untrustworthy. 
Those  worthy  of  some  degree  of  confidence  are  as  follows : 

Hiss's  Method. — In  this  method  advantage  is  taken 
of  the  fact  that  in  semisolid  nutrient  media  the  greater 
motility  of  the  typhoid  bacillus  enables  it  to  diffuse  more 
readily  through  the  medium  than  can  the  less  active 
colon  bacillus.  The  endeavor  of  Hiss  was  to  discover 
a  method  whereby  this  peculiarity  would  be  favored, 
or  at  least  not  checked,  in  the  typhoid,  and  more  or 
less  suppressed  in  the  colon  bacillus.  A  series  of  experi- 
ments demonstrated  that  if  peptone  be  omitted  and  glu- 
cose be  added  to  the  semi-solid  medium,  the  absence  of 
the  former  important  nutritive  substance  and  the  excess 
of  acidity  resulting  from  the  fermentation  of  the  latter 
had  only  slight  effect  upon  the  characteristic  develop- 
ment of  the  typhoid  bacillus  (a  diffuse  clouding  of  the 
medium),  while  the  influence  upon  the  growth  of  the 
colon  bacilli  was  to  prevent,  in  many  cases,  their  ten- 
dency to  cloud  the  medium  by  sharply  restricting  their 
growth  to  the  point  at  which  they  were  deposited.1 

1  Hiss  :  Journal  of  Experimental  Medicine,  1897,  vol.  ii.  No.  6,  p.  677. 


442  BACTERIOLOGY. 

The  composition  of  the  medium  used  is  : 

Agar-agar 5  grammes. 

Gelatin      80        " 

Liebig's  beef-extract 5        " 

Sodium  chloride 5        " 

Glucose 5-10        " 

Water 1000  c.c. 

The  gelatin  should  be  added  after  the  agar-agar  and 
other  ingredients  are  dissolved  ;  the  volume  of  the  mass 
is  then  brought  to  1000  c.c.,  and  finally  the  reaction  is 
corrected.  This  should  be  equivalent  to  a  degree  of 
acidity  that  would  require  15  c.c.  of  a  normal  sodium 
hydroxide  solution  to  the  litre  to  bring  it  to  the  phenol- 
phtalein  neutral  point. 

When  planted  as  stab-cultures  in  this  medium  and 
kept  at  body-temperature  the  growth  of  bacillus  typho- 
sus  appears  simply  as  a  diffuse  cloud,  with  little  or  no 
tendency  to  appear  more  concentrated  along  the  track  of 
the  needle  ;  while  the  development  of  the  colon  bacillus 
is  confined  to  the  neighborhood  of  the  needle-track,  is 
moderately  dense,  is  accompanied  by  the  formation  of 
gas-bubbles,  and  the  surrounding  gelatin  is  more  or 
less  clear. 

These  distinctions  were  found  by  Hiss  to  be  much 
more  marked  with  the  slowly  or  feebly  motile  speci- 
mens of  the  colon  bacillus  than  when  the  actively  motile 
varieties  were  used.  Many  of  these  latter,  except  for 
their  power  to  ferment  glucose  with  liberation  of  gas, 
were  almost,  indistinguishable  from  the  typhoid  bacillus 
in  so  far  as  their  power  to  wander  through  and  cloud 
the  medium  was  concerned.  For  the  isolation  and  dif- 
ferentiation of  colonies  of  the  two  organisms  by  the 
plate  method  the  following  medium  was  employed  : 


ISOLATING   THE  TYPHOID  BACILLUS.        443 

Agar-agar 10  grammes. 

Gelatin 25        " 

Liebig's  beef-extract 5        " 

Sodium  chloride 5        " 

Glucose 10        " 

Water 1000  c.c. 

The  reaction  of  this  medium  is  equivalent  to  2  per  cent, 
of  normal  acid  to  the  litre — i.  e.,  an  acidity  that  would 
require  20  c.c.  of  normal  sodium  hydroxide  solution  to 
the  litre  to  bring  it  to  the  phenol phtalein  neutral  point. 
In  plates  made  from  this  medium  the  deep  colonies  of 
bacillus  typliosus  are  small,  more  or  less  spherical,  and 
have  a  rough,  irregular  outline.  Their  most  character- 
istic feature  "  consists  of  well-defined,  filamentous  out- 
growths, ranging  from  a  single  thread  to  a  complete 
fringe  around  the  colony.  The  young  colonies  are  at 
times  composed  solely  of  threads."  The  fringing 
threads  grow  almost  straight  out  from  the  colonies. 
The  surface  colonies  are  small  and  have  usually  a 
dense  centre  that  is  surrounded  by  an  almost  trans- 
parent zone  or  by  a  fringe  of  threads  somewhat  similar 
to  those  seen  about  the  deeper  colonies. 

The  deep  colonies  of  the  colon  bacillus  are,  as  a  rule, 
larger,  denser,  of  an  oval  or  lens-shape,  and  are  more 
sharply  circumscribed  than  those  of  bacillus  typhosus. 
On  the  surface  they  are  also  larger,  and,  as  a  rule, 
spread  out  as  a  moderately  thick  layer  from  a  denser 
centre.  The  younger  the  colonies  of  the  typhoid  bacil- 
lus the  more  characteristic  their  appearance.  They  are 
seen  at  their  best  after  from  16  to  18  hours'  growth  at 
37.5°  C.1 

1  The  reader  is  referred  to  the  original  article  for  many  important 
details  that  are  not  included  here. 


444  BACTERIOLOGY. 

Method  of  Capaldi  and  Proskauer.1 — As  a  result  of 
an  elaborate  series  of  experiments,  these  authors  recom- 
mend the  use  of  two  speeial  culture-media  for  the  dif- 
ferentiation of  the  typhoid  and  colon  bacilli. 

Medium  No.  1  consists  of: 

Asparagine 0.2    per  cent,  in  distilled  water. 

Mannite 0.2 

Sodium  chloride 0.02 

Magnesium  sulphate    ....  0.01 

Calcium  chloride 0.02 

Mono-potassium  phosphate    .  0.2 

Medium  No.  2  consists  of: 

Witte's  peptone 2.0  per  cent,  in  distilled  water. 

Mannite 0.1 

Both  media  are  to  be  sterilized,  the  reaction  brought 
to  the  litmus  neutral  point  with  caustic  potash  solu- 
tion, and  enough  litmus  tincture  then  added  to  them  to 
cause  a  distinct,  though  not  too  intense,  purple  color. 
After  this  they  are  to  be  again  sterilized,  when  they 
are  ready  for  use. 

After  20  hours  at  37°-38°  C.  typical  colon  bacilli 
and  the  varieties  of  this  organism  develop  in  both  solu- 
tions, but  produce  add  only  in  medium  No.  1. 

The  typhoid  bacillus  grows  only  in  medium  No.  2, 
and  its  growth  is  accompanied  by  the  production  of 
an  acid  reaction. 

The  growth  of  bacillus  coli  in  medium  No.  2  is  ac- 
companied by  a  slight  alkaline  reaction. 

The  alterations  in  reaction  are  indicated  by  the  cor- 
responding changes  in  the  color  of  the  litmus  tincture 
in  the  media. 

1  Capaldi  and  Proskauer :  Zeitschrift  fur  Hygiene  und  Infektious- 
krankheiten,  1896,  Bd.  xxiii.  S.  452. 


ISOLATING   THE  TYPHOID  BACILLUS.        445 

It  is  interesting  to  note  that  in  this  test  the  usual 
reactions  produced  by  these  organisms  in  peptone  media 
containing  the  ordinary  fermentable  carbohydrates,  such 
as  glucose  and  lactose,  are  reversed. 

The  authors  state  that  this  method  has  thus  far  shown 
itself  to  be  infallible  for  the  differentiation  of  cultures 
of  typhoid  and  colon  bacilli  obtained  by  them  from 
every  available  source. 

Hunter1  recommends  the  use  of  neutral  red  as  a  dif- 
ferential test.  He  employs  it  iii  the  proportion  of  0.5 
to  1.0  c.c.  of  a  saturated  watery  solution  to  10  c.c.  of 
nutrient  agar-agar.  The  reducing  action  of  the  colon 
bacillus  causes  the  color  to  become  yellow,  while  the 
normal  red  color  is  not  affected  by  the  typhoid  bacillus. 

METHOD  OF  v.  DRIGALSKI  AND  CONRADI. — v.  Dri- 
galski  and  Conradi 2  published  a  method  for  the  detec- 
tion of  bacillus  typhosus  in  water.  In  this  method 
they  sought  to  bring  about  a  separation  of  bacillus 
typhosus  and  bacillus  coli  on  the  basis  of  their  ferment- 
ing properties.  This  they  sought  to  do  in  such  a  man- 
ner as  not  to  hinder  the  growth  of  bacillus  typhosus, 
but  rather  to  make  the  conditions  of  growth  as  favor- 
able as  possible.  Their  studies  of  the  fermentative 
properties  of  bacillus  typhosus  and  bacillus  coli  were 
carried  out  upon  the  following  carbohydrates  : 

1.  Monosaccharides :  Of  hexoses  :  glucose,  fructose, 
galactose,  mannite  and  dulcit.     Of  pentoses  :  arabinose, 
xylose,  and  rhamnose. 

2.  Disaccharides  :  Saccharose,  maltose,  lactose. 

3.  Polysaccharides  :  Amylum,  inulin,  and  dextrin. 

1  Hunter:  The  Lancet,  March  2,  1901. 

2  v.  Drigalski  and  Conradi :  Zeitschrift  fur  Hygiene,  Bd.  39,  1902, 
t  283. 


446  BA  CTERIOL  OGY. 

These  substances  were  added  to  sterile  litmus  agar  in 
the  proportion  of  1  to  100,  and  sterilization  was  again 
carried  out  for  five  to  ten  minutes  in  streaming  steam. 

In  their  studies  with  these  culture  media  they  found 
that  different  organisms  behaved  differently  with  the 
lactose.  While  colon  cultures  produce  a  red  colora- 
tion of  the  litmus  when  grown  on  the  surface  of 
the  medium,  the  typhoid  cultures  cause  no  change. 
They  also  found  that  a  culture  medium  of  greater 
density  was  of  distinct  value,  and  consequently  they 
employed  3  per  cent,  agar-agar  medium,  and  in  order 
to  overcome  the  marked  acid  production  by  the  colon 
organism  they  added  small  quantities  of  sodium  car- 
bonate. This  increase  in  the  salt  concentration  of  the 
culture  medium  did  not  bring  about  plasmolysis  of  the 
typhoid  organism.  In  order  to  make  the  conditions 
as  favorable  as  possible  for  the  growth  of  bacillus 
typhosus  they  experimented  with  a  large  number  of 
artificially  prepared  albuminous  substances.  Besides 
peptone  they  experimented  with  tropon  and  nutrose. 
The  addition  of  nutrose  brought  about  a  more  intense 
blue  coloration  by  the  typhoid  organism,  which  they 
attribute  to  the  alkali  albuminate  nature  of  the  sodium 
casein.  There  was  also  a  more  voluminous  growth  of 
the  typhoid  organism  when  they  employed  meat  infu- 
sion in  the  preparation  of  the  medium. 

Still  another  difficulty  was  encountered  in  the  identi- 
fication of  bacillus  typhosus  in  stools  because  of  colo- 
nies of  varieties  of  micrococci  also  present,  which, 
through  their  marked  acid  production,  colored  the  whole 
surface  of  the  medium.  After  a  large  number  of  nega- 
tive experiments  they  succeeded  in  finding  an  elective 
bactericidal  aniline  dye  against  the  majority  of  these 


ISOLATING    THE  TYPHOID  BACILLUS.         447 

disturbing  organisms,  which,  however,  was  not  injurious 
to  the  typhoid  and  colon  organisms.  In  the  selection 
of  an  antiseptic  coloring  substance  they  had  to  be  cer- 
tain that  the  fermentative  properties  of  the  typhoid 
bacteria  were  not  influenced  thereby.  They  experi- 
mented with  the  following  analine  dyes :  Malachite 
green,  1  to  1,000,000;  brilliant  green,  1  to  1,000,000; 
medicinal  methylene-blue,  1  to  100,000  ;  methyl  violet, 
1  to  100,000,  and  crystal  violet,  1  to  100,000.  Of 
these  the  crystal  violet  gave  the  most  satisfactory  result, 
v.  Drigalski  and  Conradi  give  the  following  direc- 
tions for  the  preparation  of  their  culture  medium  : 

a.  Preparation  of  agar :  3  pounds  of  finely  chopped 
beef  are  placed  in  two  litres  of  water  and  set  aside  for 
twenty-four  hours.     The  meat  infusion  is  boiled  for  one 
hour,  filtered,  and  20  grammes  of  Witte's  peptone,  20 
grammes  of  nutrose,  and  10  grammes  of  sodium  chloride 
are  added  and  again  boiled  for  an  hour,  filtered,  and  60 
grammes  of  agar-agar  are  added,  boiled   for  three  hours 
(or  one  hour  in  the  autoclave),  rendered  slightly  alka- 
line to  litmus-paper,  filtered,  and   boiled  for  one-half 
hour. 

b.  Litmus   solution  :    Litmus   solution   (according  to 
Kubel   and   Tiemann)  260  c.c.  boiled  ten  minutes,  add 
30  grammes  chemically   pure  lactose,  boil   fifteen  min- 
utes. 

c.  The  hot  litmus-lactose  solution  is  added  to  the  hot 
nutritive  agar,  thoroughly  mixed,  and  the  alkaline  reac- 
tion is  again  restored.     To  this  medium  is  then  added 
4  c.c.  of  a  hot  sterile  solution  of  10  per  cent,  water-free 
soda,  20  o  c.  of  freshly  prepared  solution  of  0.1  gramme 
crystal  violet  (Hochst)  in  100  c.c.  of  warm  sterile  dis- 
tilled water. 


448  BACTERIOLOGY. 

One  now  has  a  meat-infusion-peptone-nutrose-agar 
with  13  per  cent,  of  litmus  solution  and  0.01  per  thou- 
sand crystal  violet,  which  becomes  very  hard  on  solidi- 
fying, without  becoming  too  dry.  Plates  are  poured 
with  this  material  which  can  be  held  in  readiness  for 
some  time,  and  the  remainder  of  the  medium  is  placed 
in  flasks  in  portions  of  200  c.c.  each. 

If  the  lactose  is  boiled  for  a  longer  time  than  directed 
it  is  reduced  with  an  acid  reaction  of  the  culture  medium 
and  the  content  in  lactose  falls  below  the  required  quan- 
tity, and  the  alteration  in  the  color  of  the  colon  colonies 
appears  too  early.  For  this  reason  it  is  also  necessary 
to  liquefy  the  agar  as  quickly  as  possible  in  pouring 
plates  from  the  agar  medium  stored  in  flasks. 

In  employing  this  culture  medium  it  is  necessary  to 
have  a  uniform  suspension  of  a  portion  of  the  material  to 
be  examined  and  to  make  a  series  of  plate  inoculations 
from  this  suspension.  These  plate  inoculations  are  best 
made  by  means  of  a  sterile  glass  spatula. 

After  fourteen  to  sixteen  hours  at  37°  C.,  and  still 
better  after  twenty  to  twenty-four  hours,  the  cultures 
are  readily  differentiated  : 

a.  Bacillus  coli:  All  cultures  of  true  colon  that  have 
been  examined  form  colonies  of  2  to  6  or  more  millimetres 
in  diameter,  of  reddish  color  and  translucent.  In  each  intes- 
tinal evacuation  there  are  usually  several  varieties  of  colon 
colonies  which  differ  according  to  their  size  and  texture, 
translucency,  and  the  intensity  of  the  alteration  of  the 
color  which  they  bring  about.     Many  colon  colonies  are 
bright  red,  some  are  cloudy,  and  others  are  quite  opaque, 
dark-wine  red  in  color,  while  still  others  form  large  col- 
onies which  are  surrounded  by  a  red  halo. 

b.  Bacillus  typhosus:  The  colonies  have  a  diameter 


ISOLATING   THE  TYPHOID  BACILLUS.         449 

of  1  to  3  millimetres,  rarely  larger.  Their  color  is 
blue,  with  a  tendency  toward  violet.  In  structure  they 
are  glistening,  with  a  single  contour,  somewhat  of  the 
nature  of  a  dew  drop.  Only  in  isolated  instances  is  the 
colony  larger  and  more  cloudy  in  appearance. 

METHOD  OF  HOFFMANN  AND  FICKER. — Hoffrminn 
and  Picker l  have  published  a  new  method  for  the  isola- 
tion of  bacillus  typhosus  from  infected  waters,  which 
consists  in  the  addition  of  increasing  quantities  of  caf- 
fein,  crystal  violet,  and  nutrose  to  large  quantities  of  the 
water.  They  add  1  per  cent,  of  nutrose,  0.5  per  cent, 
of  caffein,  and  1  per  cent,  of  a  0.1  to  100  solution  of 
crystal  violet  to  the  water,  and  incubate  at  37°  C.  for 
twelve  to  thirteen  hours.  In  this  manner  they  reduce 
the  number  of  water  bacteria,  while  bacillus  typhosus 
increases  in  numbers.  The  three  solutions  to  be  added 
to  the  water  are  prepared  as  follows : 

1.  A  solution  of  10  grammes  nutrose  in  80  c.c.  of 
distilled  water.     The  solution  is  placed  in  a  water-bath 
for  several  hours  and  is  not  filtered. 

2.  A  solution  of  5  grammes  of  caffein  in  20  c.c.  of 
warm  (80°  C.),  sterile  distilled  water.     The  solution  is 
to  be  freshly  prepared  and  should  not  be  shaken. 

3.  A  solution  of  0.1  gramme  of  crystal  violet  in  100 
c.c.  of  sterile  distilled  water.      This   must  be  freshly 
prepared  each  time. 

900  c.c.  of  the  water  to  be  examined  are  placed  in  a 
flask  and  the  three  solutions  are  added,  and  the  mixture 
thoroughly  shaken.  After  incubation,  the  water  is  exam- 
ined according  to  several  well-known  methods.  For 
instance,  some  of  the  supernatant  portion  of  the  fluid 
is  removed  and  spread  out  in  a  thin  layer  upon  plates 

1  Hoffmann  and  Ficker  :  Hygienische  Kuudschau,  Bd.  14, 1904,  p.  1. 
29 


450  BA  CTERIOLOG  Y. 

made  with  Drigalski-Conradi  agar,  or  500  c.c.  of  the 
water  may  be  precipitated  with  typhoid  immune  serum 
according  to  the  method  of  Altschuler  and  incubated 
again  for  three  hours  and  then  plated,  or  500  c.c.  may 
be  precipitated  by  the  chemical-mechanical  method  of 
Ficker.1  The  sediment  in  the  portion  of  water  which 
has  been  precipitated  in  this  manner  is  distributed  over 
a  series  of  three  or  four  plates  of  the  Drigalski-Conradi 
medium.  Subsequently  the  sediment  is  diluted  three- 
or  fourfold  and  distributed  over  another  series  of  plates. 
By  these  methods  Hoffmann  and  Ficker  succeeded  in 
isolating  bacillus  typhosus  when  present  in  a  mixture 
of  1  :  51,867  water  bacteria. 

v.  Jaksch  and  Rau l  employed  the  method  of  Hoff- 
mann and  Ficker  for  the  isolation  of  bacillus  typhosus 
from  the  water  supply  of  the  city  of  Prague,  and  suc- 
ceeded in  finding  bacillus  typhosus  in  three  out  of  five 
samples  examined,  one  of  which  was  taken  from  a  tap 
in  the  hospital,  while  the  others  were  taken  from  the 
Moldau,  at  different  points  along  the  city  front.  The 
two  negative  samples  were  derived  from  points  along 
the  upper  portion  of  the  stream  where  there  was  less 
opportunity  for  the  water  to  become  polluted.  The 
demonstration  of  the  presence  of  bacillus  typhosus  in 
these  samples  of  water  was  substantiated  by  all  the 
known  cultural  methods  as  well  as  by  the  agglutination 
test,  using  for  the  latter  purpose  the  serum  of  a  highly 
immunized  rabbit  which  agglutinated  the  bacteria  iso- 
lated from  the  water  in  the  proportion  of  1  : 10,000. 
Higher  dilutions  were  not  made. 

1  Picker:  Hygieniscbe  Eundschau,  1904,  Bd.  xiv.,  S.  7. 

2  v.  Jaksch  and  Kau :  Centralblatt  fiir  Bacteriologie,  1904,  Bd.  xxxvi., 

S,  584. 


ISOLATING   THE  TYPHOID  BACILLUS.        451 

PRECIPITATION  METHOD  OF  FiCKER.1 — The  method 
first  proposed  by  Vallet,2  and  modified  by  Schuder,3  for 
the  demonstration  of  bacillus  typhosus  in  water  consists 
in  the  precipitation  with  sodium  hyposulphite  and  nitrate 
of  lead,  when  the  precipitate  is  dissolved  with  sodium 
hyposulphite.  This  method  was  studied  by  Ficker,  in 
the  laboratory,  by  adding  to  sterile  river  water  definite 
quantities  of  bacillus  typhosus,  but  the  results  were  not 
satisfactory.  The  experiment  showed  that  a  portion  of 
the  bacilli  were  not  carried  down  with  the  precipitate, 
while  another  portion  were  killed.  These  negative  re- 
sults led  him  to  employ,  at  the  suggestion  of  Hoffmann, 
sulphate  of  iron  as  a  precipitating  substance,  and  the 
sediment  was  dissolved  with  neutral  potassium  tartrate. 

The  method  employed  is  as  follows  :  Two  litres  of 
the  water  to  be  examined  are  placed  into  a  narrow 
sterile  glass  cylinder  and  rendered  alkaline  with  8  c.c. 
of  10  per  cent,  soda  solution,  and  afterward  7  c.c.  of  a 
10  per  cent,  sulphate  of  iron  solution  are  added  and 
mixed  with  the  water  by  means  of  a  sterile  glass  rod. 
The  cylinder  is  then  placed  in  the  ice  chest.  Precipi- 
tation is  complete  in  two  to  three  hours.  The  over- 
standing  water  is  syphoned  off,  and  the  precipitate  or 
portions  thereof  are  poured  into  sterile  test-tubes.  To 
this  precipitate  is  now  added  about  a  half  volume  of  a 
25  per  cent,  solution  of  neutral  potassium  tartrate.  The 
test-tube  is  closed  with  a  sterile  rubber  cork  and  the 
mixture  thoroughly  agitated,  whereby  the  precipitate  is 
completely  dissolved.  With  a  sterile  pipette  one  part 
of  the  mixture  is  mixed  in  a  test-tube  with  two  parts 

1  Ficker:  Hygienische  Rundschau,  1904,  Bd.  xiv.,  S.  7. 

2  Vallet:  Arch,  de  med.  exp.  et  d'anat.  path.,  1901. 

3  Schiider :  Zeitschr.  fur  Hygiene,  Bd.  xlii.,  S.  317. 


452  £A  CTERWLOG  T. 

of  sterile  bouillon,  and  this  mixture  is  distributed  over 
a  series  of  Drigalski-Conradi  plates.  Ficker  advises 
when  possible  the  use  of  a  centrifuge  for  the  separation 
of  the  precipitate,  as  he  believes  the  results  are  likely 
to  be  more  satisfactory. 

ISOLATION  OF  BACILLUS  TYPHOSUS  FROM  CADA- 
VERS.— The  spleen  of  a  patient  dead  of  typhoid  fever  is 
the  most  reliable  source  from  which  to  obtain  cultures 
of  the  typhoid  bacillus  for  study.  But  it  must  always  be 
remembered  that  the  same  channels  through  which  the 
typhoid  bacillus  gains  access  to  this  viscus  are  likewise 
open  to  other  organisms  present  in  the  intestines,  and 
for  this  reason  bacillus  coli,  a  normal  inhabitant  of  the 
colon,  may  also  be  found  in  this  locality. 

NOTE. — Obtain  a  pure  culture  of  typhoid  bacilli,  and 
from  this  make  inoculations  upon  a  series  of  potatoes 
of  different  ages  and  from  different  sources.  Do  they 
all  grow  alike  ? 

Before  sterilizing  render  another  lot  of  potatoes  slightly 
acid  with  a  few  drops  of  very  dilute  acetic  acid ;  render 
others  very  slightly  alkaline  with  dilute  caustic  soda. 
Are  any  differences  in  the  growths  noticeable  ? 

Make  a  series  of  twelve  tubes  of  peptone  solution  to 
which  rosolic  acid  has  been  added.  Inoculate  them  all 
with  as  nearly  the  same  amount  of  material  as  possible 
(one  loopful  from  a  bouillon  culture  into  each  tube) ; 
place  them  all  in  the  incubator.  Is  the  color-change, 
as  compared  with  that  of  the  control-tube,  the  same  in 
all  cases. 

Compare  the  morphology  of  cultures  of  the  same  age 
on  gelatin,  agar-agar,  and  potato. 

Select  a  culture  in  which  the  vacuolations  are  quite 


BACILLUS  COLT.  453 

marked.  Examine  this  culture  unstained.  Do  the 
organisms  look  as  if  they  contained  spores?  How 
would  you  demonstrate  that  the  vacuolations  are  not 
spores  ?  What  is  the  crucial  test  for  spores  ? 

Obtain  from  normal  faeces  a  pure  culture  of  the  com- 
monest organism  present.  Write  a  full  description  of 
it.  Now  make  parallel  cultures  of  this  organism  and 
of  the  typhoid  bacillus  on  all  the  different  media  ?  How 
do  they  differ?  In  what  respects  are  they  similar? 

BACILLUS    COLI    (ESCHERICH),    MIGULA,    1900. 

Synonyms:  Neapeler  bacillus,  Emmerich,  1884;  Bacillus  pyogenes 
foptidus,  Passet,  1885;  Emmerich's  bacillus,  Eisenberg,  1886;  Bac- 
terium coli  commune,  Escherich,  1886. 

This  organism  was  discovered  by  Escherich,  in  1886, 
in  the  intestinal  discharges  of  milk-fed  infants.  It  has 
since  been  demonstrated  to  be  a  normal  inhabitant  of  the 
intestines  of  man  and  of  certain  domestic  animals 
(bovines,  hogs,  dogs). 

For  a  time  after  its  discovery  it  was  considered  of 
but  little  importance  and  attracted  attention  only  be- 
cause of  its  resemblance,  in  certain  respects,  to  the  bacil- 
lus of  typhoid  fever,  with  which  it  was  occasionally 
confounded.  In  this  particular  it  still  serves  as  a 
subject  for  study.  Some  have  even  gone  so  far  as  to 
regard  them  as  but  varieties  of  one  and  the  same 
species,  though  in  the  present  state  of  our  knowledge 
this  is  an  assumption  for  which  as  yet  there  are 
not  sufficient  grounds.  That  they  possess  in  common 
certain  general  points  of  resemblance  and  often  ap- 
proach one  another  in  some  of  their  biological  peculiar- 
ities is  true ;  but,  as  we  shall  learn,  they  each  possess 
peculiarities  which,  when  considered  together,  render 


454  BACTERIOLOGY. 

their  differentiation  from  one  another  a  matter  of  but 
little  difficulty. 

With  the  wider  application  of  bacteriological  methods 
to  the  study  of  pathological  processes  it  was  occasion- 
ally observed  that,  under  favorable  circumstances, 
bacillus  coll  disseminated  from  its  normal  habitat 
and  appeared  in  remote  organs,  often  associated  witu 
diseased  conditions.  This  was  at  first  considered 
of  but  little  importance,  and  its  presence  in  these 
localities  was  usually  regarded  as  accidental.  Its 
repeated  appearance,  however,  in  different  parts  of 
the  body  outside  of  the  intestines,  and  the  frequency 
of  its  association  with  pathological  conditions,  ultimately 
attracted  attention  to  it,  and  in  consequence  during  the 
past  few  years  a  great  deal  has  been  written  concerning 
the  possible  pathogenic  nature  of  this  organism. 

The  fact  that  it  is  a  commensal  species,  always  inti- 
mately associated  with  certain  of  our  life-processes, 
together  with  the  fact  that  it  is  known  to  appear  in 
organs  other  than  that  in  which  it  is  normally  located, 
and  that  its  occurrence  in  diseased  conditions  is  not 
rare,  justifies  the  opinion  that  it  is  one  of  the  most 
important  of  the  micro-organisms  with  which  we  have 
to  deal. 

While  not  generally  considered  a  pathogenic  organ- 
ism, there  is,  nevertheless,  sufficient  evidence  to  war- 
rant the  statement  that  under  favorable  conditions  of 
reduced  vitality  on  the  part  of  the  animal  tissues,  this 
organism  may  assume  pathogenic  properties,  so  that  its 
presence  in  diseased  conditions  is  not  always  to  be  con- 
sidered as  accidental,  though  this  is  frequently  the  case. 

The  morphological  and  cultural  peculiarities  of  bacillus 
coli  are  as  follows  : 


BACILLUS  COLL  455 

MORPHOLOGY. — In  shape  it  is  a  rod  with  rounded 
ends,  sometimes  so  short  as  to  appear  almost  spherical, 
while  again  it  is  seen  as  very  much  longer  threads. 
Often  both  forms  are  associated  in  the  same  culture. 
It  may  occur  as  single  cells,  or  as  pairs  joined  end  to 
end. 

It  has  no  peculiar  morphological  features  that  can 
aid  in  its  identification,  for  in  this  respect  it  simu- 
lates many  other  organisms.  It  is  usually  said  to  be 
motile,  and  undoubtedly  is  motile  in  the  majority  of 
cases ;  but  its  movements  are  at  time  so  sluggish  that  a 
positive  opinion  is  often  difficult. 

By  Loffler's  method  of  staining,  flagella  can  be  de- 
monstrated, though  usually  not  in  such  numbers  as  are 
seen  to  occur  on  the  typhoid  fever  bacillus. 

It  does  not  form  spores. 

It  grows  both  with  and  without  free  oxygen. 

Ox  GELATIN. — On  the  surface  its  colonies  appear  as 
small,  dry,  irregular,  flat,  blue- white  points  that  are 
commonly  somewhat  dentated  or  notched  at  the  margin. 
They  are  a  trifle  denser  at  the  centre  than  at  the 
periphery,  and  are  often  marked  at  or  near  the  middle 
by  an  oval  or  round  nucleus-like  mass — the  original 
colony  from  which  the  layer  on  the  surface  developed. 
When  located  in  the  depths  of  the  gelatin,  and  ex- 
amined with  a  low-power  lens,  they  are  at  first  seen  to 
be  finely  granular  and  of  a  very  pale  greenish-yellow 
color;  later  they  become  denser,  darker,  and  much 
more  markedly  granular;  in  shape  they  are  round, 
oval,  and  lozenge-like.  AVhen  the  surface  colonies  are 
viewed  under  a  low  power  of  the  microscope  they  pre- 
sent essentially  the  same  appearance  as  that  given  for 
the  colonies  of  the  bacillus  of  typhoid  fever,  viz.,  they 


456  BACTERIOLOGY. 

resemble  flattened  pellicles  of  glass-wool,  or  patches  of 
finely  ground  colorless  glass.  Colonies  of  this  organ- 
ism on  gelatin  are  frequently  encountered  that  cannot 
be  distinguished  from  those  resulting  from  the  growth 
of  bacillus  typhosus  ;  although,  as  a  rule,  their  growth  is 
a  little  more  luxuriant. 

In  stab-  and  smear-cultures  on  gelatin  the  surface- 
growth  is  flat,  dry,  and  blue- white  or  pearl  color. 
Limited  growth  occurs  along  the  track  of  the  needle  in 
the  depths  of  the  gelatin.  As  the  culture  becomes 
older  the  gelatin  round  about  the  surface-growth  may 
gradually  lose  its  transparency  and  become  cloudy, 
often  quite  opaque.  In  still  older  cultures  small  root- 
or  branch-like  projections  from  the  surface-growth  into 
the  gelatin  are  sometimes  seen.  At  times  these  may  be 
of  a  distinctly  crystalline  appearance. 

It  does  not  cause  liquefaction  of  gelatin. 

Its  growth  on  nutrient  agar-agar  and  on  blood- serum 
is  luxuriant,  but  not  characteristic. 

In  bouillon  it  causes  diffuse  clouding  with  sedimen- 
tation. In  some  bouillon  cultures  an  attempt  at  pel- 
licle-formation on  the  surface  may  be  seen,  but  this  is  ex- 
ceptional. In  old  bouillon  cultures  the  reaction  becomes 
alkaline  and  a  decided  fsecal  odor  may  be  detected. 

Its  growth  on  potato  is  rapid  and  voluminous,  ap- 
pearing after  twenty-four  to  thirty-six  hours  in  the 
incubator  as  a  more  or  less  tabulated  layer  of  a  drab, 
dark-cream,  or  brownish-yellow  color. 

In  neutral  milk  containing  a,  little  litmus  tincture 
the  blue  color  is  changed  to  red  after  from  eighteen  to 
twenty-four  hours  in  the  incubator,  and,  in  addition, 
the  majority  of  cultures  cause  firm  coagulation  of  the 
casein  in  about  thirty-six  hours,  though  frequently  this 


BACILLUS  COLL  457 

takes  longer.  Very  rarely  the  litmus  may  indicate  the 
production  of  acid  and  no  coagulation  occur. 

In  media  containing  glucose  it  grows  rapidly  and 
causes  active  fermentation,  with  liberation  of  carbonic 
acid  and  hydrogen.  If  cultivated  in  solid  media  to 
which  glucose  (2  per  cent.)  has  been  added,  the  gas- 
formation  is  recognized  by  the  appearance  of  numerous 
bubbles  along  and  about  the  points  of  growth.  If  cul- 
tivated in  fluid  media,  also  containing  glucose,  in  the 
fermentation-tube,  evidence  of  fermentation  is  given  by 
the  collection  of  gas  in  the  closed  arm  of  the  tube. 

On  lactose-litmus-agar-agar  its  colonies  are  pink  and 
the  color  of  the  surrounding  medium  is  changed  from 
blue  to  red. 

It  produces  indol  in  both  bouillon  and  peptone  solu- 
tion. 

In  Dunham's  peptone  solution  it  produces  indol  in 
from  forty-eight  to  seventy-two  hours. 

It  stains  with  the  ordinary  aniline  dyes.  It  is  decol- 
orized when  treated  by  the  method  of  Gram. 

By  comparing  what  has  been  said  of  bacillus  typho- 
sus  and  of  bacillus  coli  it  will  be  seen  that,  while  they 
simulate  each  other  in  certain  respects,  they  nevertheless 
possess  individual  characteristics  by  which  they  may 
readily  be  differentiated.  The  least  variable  of  the  dif- 
ferential points  are : 

1.  Motility  of   bacillus  typhosus  is  much  more  con- 
spicuous, as  a  rule,  than  is  that  of  bacillus  coli. 

2.  On  gelatin,  colonies  of  the  typhoid  bacillus  de- 
velop more  slowly  than  do  those  of  the  colon  bacillus. 

3.  On  potato,  the  growth  of  the  typhoid  bacillus  is 
usually  invisible  (though  not  always) ;  while  that  of  the 
colon  bacillus  is  rapid,  luxuriant,  and  always  visible. 

4.  The  tvphoid  bacillus  does  not  cause  coagulation  of 


458  BACTERIOLOGY. 

milk  with  acid  reaction      The  colon  bacillus  does  this 
in  from  thirty-six  to  foi  y-eight  hours  in  the  incubator. 

5.  The   typhoid    bac  Jus  never  causes    fermentation, 
with  liberation  of  gas,  _n  media  containing  glucose,  lac- 
tose, or  saccharose.     The  colon  bacillus   is  conspicuous 
for  its  power  of  causing  gaseous  fermentation  in  such 
solutions. 

6.  In  nutrient  agar-agar  or  gelatin  containing  lactose 
and  litmus  tincture,  and  of  a  slightly  alkaline  reaction, 
the  color  of  the  colonies  of  typhoid  bacillus  is  pale  blue, 
and  there  is  no  reddening  of  the  surrounding  medium ; 
while  colonies  of  the  colon  bacillus  are  pink  and  the 
medium  round  about  them  becomes  red. 

7.  The  typhoid  bacillus  does  not,  as  a  rule,  possess  the 
property  of  producing  indol  in  solutions  of  peptone ;  the 
growth  of  the  colon  bacillus  in  these  solutions  is  accom- 
panied by  the  production  of  indol  in  from  forty-eight 
to  seventy-two  hours  at  37°  to  38°  C. 

ANIMAL  INOCULATIONS. — As  with  the  bacillus  of 
typhoid  fever,  the  results  of  inoculation  of  animals  with 
cultures  of  this  organism  cannot  be  safely  predicted. 
According  to  the  observations  of  Escherich,  Emmerich, 
Weisser,  and  others,  the  effects  that  do  appear  are  in 
most  instances  to  be  attributed  to  the  toxic  rather  than 
to  the  infective  properties  of  the  culture  used. 

When  introduced  into  the  subcutaneous  tissues  of 
mice  it  has  no  effect,  while  similar  inoculations  of  guinea- 
pigs  are  sometimes  (not  always)  followed  by  abscess- 
formation  at  the  point  of  operation,  or  by  alterations  very 
similar  to  those  produced  by  intravascular  inoculation, 
viz.,  death  in  less  than  twenty-four  hours,  accompanied 
by  redness  of  the  peritoneum  and  marked  hypersemia 
and  ecchymoses  of  the  small  intestine,  together  with 


BACILLUS  COLT.  459 

swelling  of  Peyer's  patches.  The  caecum  and  colon 
may  remain  unchanged  or  present  enlarged  follicles. 
There  may  or  may  not  be  an  accumulation  of  fluid  in 
the  abdominal  cavity ;  but  peritonitis  is  rarely  present. 
The  small  intestine  may  contain  bloody  mucus. 

Intravenous  inoculation  of  rabbits  may  be  followed 
by  similar  changes,  with  often  the  occurrence  of  diar- 
rhoea before  death,  which  may,  in  the  acute  cases,  result 
in  from  three  to  forty  hours.  In  another  group  of 
cases  acute  fatal  intoxication  does  not  result,  and  the 
animal  lives  for  weeks  or  months,  dying  ultimately  of 
what  appears  to  be  the  effects  of  a  slow  or  chronic  form 
of  infection.  For  a  few  hours  after  inoculation  these 
animals  present  no  marked  symptoms ;  exceptionally, 
somnolence  and  diarrhoea  have  been  observed  at  this 
period,  indicating  acute  intoxication  from  which  the 
animal  has  recovered.  The  affection  is  unattended  by 
fever.  The  most  marked  symptom  is  loss  of  weight. 
This  is  usually  progressive  from  the  first  or  second  day 
after  inoculation,  with  slight  fluctuations  until  death. 

At  autopsy  the  animal  is  found  to  be  emaciated. 
The  subcutaneous  tissues  and  the  muscles  appear  pale 
and  dry.  The  serous  cavities,  particularly  the  pericar- 
dial,  may  contain  an  excess  of  serum.  The  viscera  are 
anaemic.  The  spleen  is  small,  thin,  and  pale.  Ex- 
ceptionally ulcers  and  ecchymoses  are  observed  in  the 
caecum,  but  generally  there  are  no  lesions  of  the  intes- 
tinal tract. 

The  most  striking  and  constant  lesions,  those  most 
characteristic  of  the  affection,  are  in  the  bile  and  in  the 
liver ;  in  some  cases  the  quantity  of  bile  may  not  exceed 
the  normal,  but  in  others  the  gall-bladder  may  be  ab- 
normally distended  with  bile.  The  bile  is  nearly  color- 


460  BACTERIOLOGY. 


less  or  has  a  pale  yellowish  or  brownish  tint,  with  little 
or  no  greenish  color.  Its  consistence  is  much  less 
viscid  than  normal,  being  often  thin  and  watery.  It 
usually  contains  small,  opaque,  yellowish  particles  or 
clumps  which  can  be  seen  floating  in  it,  even  through  the 
walls  of  the  gall-bladder.  These  clumps  consist  micro- 
scopically of  bile-stained,  apparently  necrotic,  epithelial 
cells ;  leucocytes  in  small  numbers ;  amorphous  masses 
of  bile-pigment,  and  bacteria  often  in  zoogloea-like 
clumps.  Similar  material  is  found  in  the  larger  bile- 
ducts. 

The  liver  frequently  contains  opaque,  whitish  or  yel- 
lowish-white spots  and  streaks  of  irregular  size  and 
shape,  which  give  a  peculiar  mottling  to  the  organ  when 
present  in  large  number.  These  areas  may  be  numer- 
ous, or  only  one  or  two  may  be  found.  In  size  they 
range  from  minute  points  to  areas  of  from  2  to  3  cm.  in 
extent.  By  microscopic  examination  they  are  found  to 
represent  localities  where  the  liver-cells  have  undergone 
necrosis  accompanied  by  emigration  of  leucocytes,  and 
the  cells  about  them  are  in  a  condition  of  fatty  degenera- 
tion. In  sections  of  the  liver  masses  of  the  bacilli  may 
be  discovered  in  and  about  the  necrotic  foci  just  de- 
scribed. 

At  these  autopsies  the  colon  bacillus  is  not  found 
generally  distributed  through  the  body,  but  is  only  to  be 
detected  in  the  bile,  liver,  and  occasionally  in  the  spleen.1 

BACILLUS    PARATYPHOSUS. 

During  the  past  five  years  the  careful  bacteriological 
examination  of  cases  of  continued  fever  in  which  the 

1  Consult  paper  by  Blachstein  on  this  subject,  Johns  Hopkins  Hos- 
pital Bulletin,  1891,  vol.  ii.,  p.  96. 


BACILLUS  PAMATTPHO8US.  461 

agglutination  reaction  with  the  typhoid  bacillus  was 
absent,  has  revealed  a  group  of  bacilli  which  differ  from 
bacillus  typhosus  in  certain  important  particulars.  These 
bacteria  possess  characters  which  are  intermediate  be- 
tween those  of  bacillus  typhosus  and  bacillus  coli,  some 
resembling  more  closely  bacillus  typhosus,  and  others 
bacillus  coli,  and  for  these  reasons  they  have  sometimes 
been  classed  as  the  intermediate  group.  Some  of  the 
organisms  isolated  from  such  cases  of  continued  fever 

o 

resemble  very  closely  bacillus  enteriditis,  which  Gaertner 
found  in  cases  of  meat  poisoning. 

The  general  opinion  to-day  is  that  these  organisms 
produce  a  form  of  infection  sometimes  resembling  in 
many  of  its  characters  that  produced  by  bacillus  ty- 
phosus. The  infection,  however,  is  usually  of  a  milder 
type  and  only  a  comparatively  small  number. of  cases 
have  terminated  fatally,  so  that  the  pathology  of  the 
disease  is  not  well  known.  Moreover,  the  biological  char- 
acters of  the  different  organisms  isolated  from  cases  of 
paratyphoid  fever  show  such  wide  variations  that  it  is 
probable  that  the  pathology  of  different  cases  also 
varies  with  the  particular  type  of  organism  causing  the 
infection. 

Buxton l  was  one  of  the  first  to  make  a  careful  com- 
parative study  of  the  morphology  and  biology  of  this 
group  of  organisms.  He  classifies  the  intermediary 
group  of  organisms  in  the  following  manner : 

"  Paracolons :  those  which  do  not  cause  typhoidal 
symptoms  in  man.  A  group  containing  numerous  dif- 
ferent members,  but  culturally  alike. 

"  Paratyphoids  :  those  which  cause  typhoidal  symp- 
toms. 

1  Buxton:  Journal  of  Medical  Research,  1902,  vol.  viii.,  p.  201. 


462  B  A  CTERIOL  OGY. 

"(a)  A  distinct  species  culturally  unlike  the  para- 
colons. 

"  (6)  A  distinct  species  culturally  resembling  the 
paracolons." 

JBuxton  acknowledges,  as  has  also  been  found  by 
others,  that  some  of  those  producing  typhoidal  symp- 
toms cannot  be  distinguished  culturally  from  some 
members  of  the  paracolon  group.  Morphologically,  the 
intermediates  cannot  be  distinguished  with  certainty  from 
each  other,  nor  from  bacillus  typhosus  or  bacillus  coli. 
All  the  organisms  of  the  intermediate  group  have  the 
morphological  characters  of  the  colon-typhoid  group  of 
organisms. 

The  biological  differences  on  agar-agar,  blood  serum, 
gelatin,  and  bouillon,  between  the  members  of  the  inter- 
mediate group,  and  between  bacillus  typhosus  and  bacil- 
lus coli  are  too  insignificant  and  uncertain  to  be  of  any 
assistance  in  a  differentiation  between  members  of  the 
group.  In  litmus  milk  certain  well-marked  differences 
between  different  members  of  the  group  are  noticed. 
None  of  the  organisms  of  the  intermediate  group  pro- 
duce coagulation.  Some  produce  a  slight  initial  acidity, 
which  is  later  followed  by  an  alkaline  reaction.  Still 
other  members  of  the  group  produce  an  acidity  amount- 
ing to  1  per  cent. 

Buxton  states  that  the  intermediates  can  be  distin- 
guished from  bacillus  typhosus  by  their  power  of  fer- 
menting the  disaccharid  maltose  and  all  the  monosac- 
charids  with  gas  formation.  On  the  other  hand  they 
can  be  distinguished  from  bacillus  coli  by  their  inability 
to  form  acid  and  gas  in  lactose  media. 

The  agglutination  reaction  of  members  of  the  inter- 
mediate group  with  the  serum  of  an  animal  immunized 


BACILLUS  PARATYPHOSUS.  463 

with  one  of  the  organisms  varies  with  the  different 
organisms.  The  more  closely  a  member  of  the  group 
resembles  culturally  the  organism  employed  in  immuni- 
zing the  animal  the  more  readily  is  it  agglutinated.  In 
attempts  to  diagnose  paratyphoid  infection  it  is  well  to 
bear  this  fact  in  mind  and  make  agglutination  tests  with 
different  members  of  the  group  and  the  blood  of  the 
patient. 


CHAPTER   XXL 

BACILLUS    DYSENTER1.E. 

The  group  of  bacilli  found  in  cases  of  epidemic,  endemic,  and  sporadic 
dysentery — The  morphological,  biological,  and  pathogenical  char- 
acters of  the  several  members  of  the  group — The  differentiation 
of  the  different  types  of  bacilli. 

THE  investigations  of  epidemic  dysentery  by  Shiga, 
Flexner,  Kruse,  Vedder,  Duval,  Basset,  Park,  and 
many  others,  have  demonstrated  that  this  disease  is 
caused  by  an  organism  that  varies  somewhat  in  its  char- 
acters as  encountered  in  different  cases.  So  far  at  least 
four  distinct  types  of  the  organism  have  been  found  that 
differ  in  minor  particulars,  though  not  sufficiently  to  war- 
rant their  separation  from  each  other  into  distinct  species. 
The  type  of  organism  first  encountered  by  Shiga  in 
Japan  is  the  one  that  is  probably  very  widely  distrib- 
uted because  it  has  been  found  in  practically  every  place 
where  investigations  have  been  made.  The  type  of 
organism  encountered  by  Flexner  in  his  investigations 
in.  the  Philippine  Islands,  has  also  been  found  very  gen- 
erally in  the  United  States,  especially  in  dysentery 
occurring  in  infants.  The  type  of  organism  isolated  by 
Hiss  and  Russell,  and  later  by  Park  and  his  associates, 
has  most  of  the  characteristics  of  the  Flexner  type  of 
organism,  though  the  agglutination  reaction  shows  that 
it  is  not  identical  with  it. 

At  first  the  German  investigators  were  inclined  to 
regard  the  Flexner  type  of  organism  as  having  no  caus- 
ative relation  whatever  to  dysentery,  but  the  later  de- 

464 


BACILLUS  DYSENTERIC.  465 

tailed  studies  all  strengthen  the  assumption  that  the 
Shiga  type  of  the  organism  is  not  the  only  one  concerned 
in  causing  epidemic  dysentery.  In  a  number  of  cases 
of  dysentery  two,  and  at  times  three,  types  of  bacillus 
dysenteric  have  been  encountered.  Thus  far  it  has  been 
impossible  to  differentiate  clinically  between  the  infections 
produced  by  the  one  or  the  other  type  of  organism. 
Both  severe  and  mild  cases  have  been  shown  to  be  in- 
fected with  either  type,  and  the  amount  of  blood  and 
mucus  in  the  stools  appears  to  be  the  same  in  infection 
with  each  type  of  organism. 

THE  SHIGA  TYPE  OF  ORGANISM. — The  evidence 
presented  by  Shiga,  who  discovered  this  organism  in 
1898,  in  Japan,  and  the  subsequent  observations  of 
Flexner  upon  dysentery  in  the  Philippine  Islands, 
leaves  little  room  for  doubt  that,  in  so  far  as  acute 
epidemic  dysentery  is  concerned,  the  organism  under 
consideration  may  reasonably  be  regarded  as  the  causa- 
tive factor.  By  both  Shiga  and  Flexner  the  organism 
was  almost  uniformly  encountered  in  the  intestinal  con- 
tents, the  intestinal  walls,  and  the  mesenteric  glands 
during  the  acute  stages  of  the  disease.  Later  it  was 
frequently  missed,  and  this  became  more  common  as  the 
malady  progressed  to  chronicity  or  recovery. 

It  is  a  bacillus  of  medium  size,  with  rounded  ends. 
In  general  its  morphology  may  properly  be  likened  to 
that  of  either  the  typhoid  or  colon  bacillus. 

It  is  motile  and  does  not  form  spores. 

It  can  be  stained  with  any  of  the  ordinary  aniline 
dyes.  It  is  decolorized  by  the  method  of  Gram.  It 
may  be  cultivated  on  all  the  ordinary  media.  It  grows 
at  room-temperature,  but  better  at  the  temperature  of 
the  body.  It  does  not  liquefy  gelatin. 

30 


466  BACTERIOLOGY. 

The  colonies  upon  agar-agar  present  nothing  charac- 
teristic ;  those  on  gelatin  are  at  first — /.  e.,  just  after  iso- 
lation from  the  body — like  those  of  bacillus  typhosus ; 
later  on,  after  the  organism  has  been  kept  under  condi- 
tions of  continuous  saprophytic  growth,  the  colonies  may 
be  thicker,  denser,  moister,  and  less  translucent,  but 
always  suggesting  the  peculiar,  leaf-like  contour  char- 
acteristic of  the  colonies  of  the  colon-typhoid  group 
under  similar  conditions.  In  gelatin  stab-cultures  there 
is  growth  along  the  track  made  by  the  needle,  and  little 
tendency  to  lateral  development  over  the  surface. 

On  potato,  its  growth  may  be  so  limited  as  to  be 
scarcely  visible,  or  it  may  appear  as  a  moderately  volu- 
minous grayish-brown  or  light-brown  layer  along  the 
track  made  by  the  needle,  and  spreading  laterally  be- 
yond this.  Between  these  extremes  all  gradations  may 
be  seen  according  to  the  suitability  of  the  potato  used. 

In  bouillon  it  causes  uniform  clouding  and  a  more  or 
less  dense  sediment.  It  does  not  form  a  pellicle. 

Growth  on  blood-serum  is  not  accompanied  by  lique- 
faction (digestion). 

Glycerin  agar-agar  appears  less  suited  to  its  growth 
than  plain  nutrient  agar-agar. 

It  does  not  ferment  either  glucose,  saccharose,  or  lac- 
tose, with  liberation  of  gas ;  although  in  glucose  media 
there  is  a  slight  increase  of  acidity. 

When  grown  in  litmus-milk,  the  latter  after  twenty- 
four  to  seventy-two  hours  at  body-temperature  becomes 
a  pale  lilac.  Later  on — i.  e.,  after  six  to  eight  days — 
there  is  a  development  of  alkali,  and  the  lilac  tint  gives 
way  to  a  deep,  distinct  blue  color.  Coagulation  is  never 
observed. 

It  is  either  incapable  of  producing  indol,  or  has  this 


BACILLUS  DYSENTERIC.  467 

faculty  to  so  limited  a  degree  as  to  make  the  matter 
doubtful. 

When  mixed  with  blood-serum  of  individuals  suffer- 
ing from  this  form  of  dysentery  a  positive  agglutination 
reaction  is  often  obtained. 

It  is  pathogenic  by  both  subcutaneous  and  intraperi- 
toneal  inoculation  for  the  ordinary  laboratory  test-ani- 
nuils — i.  e.,  mice,  guinea-pigs,  and  rabbits. 

When  injection  is  made  beneath  the  skin,  death  results 
in  from  two  to  four  days,  according  to  the  dose  and  viru- 
lence of  the  culture  used. 

The  most  striking  lesion  is  that  observed  at  and  about 
the  site  of  inoculation.  This  consists  of  oedema,  hem- 
orrhagic  exudation,  and,  in  delayed  cases,  more  or  less 
of  pus  formation.  The  subcutaneous  lymph-glands  are 
often  enlarged  and  reddened,  and  a  serous  exudation  is 
frequently  encountered  in  the  great  serous  cavities.  Of 
the  animals  mentioned,  the  rabbit  is  most  apt  to  survive 
the  subcutaneous  inoculation. 

When  injected  into  the  peritoneal  cavity,  death  takes 
place  in  from  a  few  hours  to  five  or  six  days,  according 
to  dose  and  virulence  of  the  culture  used. 

At  autopsy  the  superficial  lymph-glands  are  enlarged 
and  reddened ;  the  peritoneum  contains  more  or  less  of 
turbid  fluid  and  small  masses  of  leucocytes ;  the  pleural 
and  pericardia!  cavities  may  contain  clear  fluid ;  the 
spleen  is  swollen  ;  the  adrenals  and  kidneys  are  con- 
gested ;  there  may  be  a  grayish  exudate  over  the  liver, 
spleen,  and  intestines,  the  bloodvessels  are  injected  ;  the 
small  intestine  may  be  filled  with  semifluid  or  fluid  mat- 
ter ;  there  may  be  ecchymosis  of  the  intestinal  mucosa, 
and  Peyer's  patches  may  be  enlarged  and  reddened. 

The   distribution   of    the   bacilli   varies:    sometimes 


468  BACTERIOLOGY. 

there  is  a  general  invasion  of  the  body  by  the  bacilli ; 
at  others  they  are  only  to  be  found  at  the  local  site  of 
inoculation.  Sometimes  they  can  be  detected  in  the 
intestinal  contents  after  both  subcutaneous  and  intra- 
peritoneal  inoculation;  at  other  times  they  cannot. 

If  the  stomach  contents  be  neutralized  and  large  doses 
of  the  bacilli  be  administered  per  os,  death  may  occur. 
Under  these  conditions  the  small  intestine  is  hypersemic 
and  contains  blood-stained  mucoid  matter,  from  which 
the  bacilli  may  usually  be  cultivated. 

If  cultures  be  fed  to  cats  after  administration  of 
croton  oil,  a  fatal  diarrhoea  may  ensue.  The  mucous 
membrane  of  the  large  intestine  is  injected,  its  surface 
covered  with  mucus,  and  its  contents  mucoid.  From  the 
latter  the  bacilli  may  be  recovered  in  culture. 

A  fatal  diarrhoea  may  follow  the  simple  feeding  of 
cultures  to  dogs.  This  occurs  in  somewhat  less  than 
six  days.  The  condition  of  the  contents  and  walls  of 
the  large  intestine  is  essentially  similar  to  that  seen  in 
the  cat. 

In  view  of  the  fact  that  marked  evidences  of  intoxi- 
cation may  follow  upon  the  injection  of  suspensions  of 
dead  cultures  of  this  organism  (solid  cultures  killed  by 
exposure  to  60°  C.),  it  is  probable  that  the  pathogen- 
icity  of  this  organism  is  referable  to  the  poisonous  nature 
of  the  proteid  making  up  the  bodies  of  the  bacilli,  rather 
than  to  a  soluble  intoxicant  secreted  or  manufactured  by 
them  in  the  course  of  their  growth. 

THE  HISS-RUSSELL  TYPE  OF  ORGANISM. — In  the 
detailed  study  of  dysentery  and  summer  diarrhoea  in 
infants,  which  has  been  in  progress  for  several  years, 
a  type  of  bacillus  dysenterise  has  been  encountered 
which  has  the  property  of  fermenting  mannite  as  well 


BACILLUS  DYSENTERIC.  469 

as  dextrose.  The  Shiga  type  ferments  dextrose,  but 
none  of  the  other  carbohydrates. 

THE  STRONG  TYPE  OF  ORGANISM. — This  type  of 
organism  has  many  of  the  characters  of  the  Harris  type, 
though  it  ferments  only  mannite,  dextrose,  and  saccha- 
rose. 

THE  HARRIS  TYPE  OF  ORGANISM. — This  type  of 
bacillus  dysenterise  was  first  encountered  by  Strong 
while  working  in  the  Philippine  Islands.  It  has  since 
been  encountered  quite  frequently  in  the  United  States, 
especially  in  the  summer  diarrhoeas  in  infants.  This 
organism  ferments  mannite  as  well  as  dextrose,  maltose, 
saccharose,  and  dextrin. 

It  is  only  by  careful  observations  of  the  reactions 
with  the  different  carbohydrates  that  it  is  possible  to 
differentiate  between  these  different  types  of  bacillus 
dysenteriae,  as  has  been  shown  by  Hiss  1  and  by  others. 

THE  AGGLUTINABILITY  OF  BACILLUS  DYSENTERIC. 
—The  study  of  the  influence  of  the  agglutinins  in  dys- 
entery immune  serum  has  also  served  to  differentiate 
between  different  types,  of  bacillus  dysenterise.  Normal 
serums,  especially  those  of  bovines  and  of  goats,  also 
yield  very  instructive  results.  These  variations  in  the 
agglutinability  of  the  several  types  of  bacillus  dysenterise, 
especially  in  normal  serums,  were  first  pointed  out  by 
Bergey,2  and  have  since  been  substantiated  by  many 
other  investigators  (see  especially  Park  and  Hiss,  loo.  ciL). 

The  different  types  of  bacillus  dysenterise  can  easily 
be  distinguished  by  their  relative  agglutinability.  In 
order  to  bring  out  the  relative  influence  of  the  immune 
serum  upon  each  variety  of  bacillus  dysenterise  it  is 

1  Hiss :  Journal  of  Medical  Research,  vol.  viii.,  Dec.,  1904. 

2  Bergey:  Journal  of  Medical  Research,  1903,  vol.  v,,  p.  21. 


470  BACTERIOLOGY. 

necessary  to  test  carefully  the  limits  of  its  agglutinating 
power  for  each  variety.  When  this  is  done  it  will  be 
found  that  the  serum  of  an  animal  immunized  with  the 
Shiga  type  of  organism  will  agglutinate  that  type  of 
organism  in  high  dilutions,  say  1  :  5000,  while  the 
Harris  type  of  organism  will  only  be  agglutinated  in 
dilutions  of  1  :  200,  and  the  Hiss-Russell  type  of  organ- 
ism in  dilutions  of  1  :  50.  On  the  other  hand,  the  serum 
of  an  animal  immunized  with  the  Flexner  type  of  or- 
ganism will  agglutinate  that  type  of  organism  in  high 
dilutions,  say  1  : 10,000,  while  the  other  two  types  of  the 
organism  will  be  agglutinated  only  in  dilutions  of  1  : 1 00. 
The  serum  of  an  animal  immunized  with  the  Hiss- 
Russell  type  of  organism  will  agglutinate  that  type  of 
organism  in  dilutions,  say  of  1  :  1000,  while  the  Harris 
type  is  agglutinated  only  in  dilutions  of  1  : 100,  and  the 
Shiga  type  in  dilutions  of  1  :  20. 

PROTECTIVE  INOCULATION. — By  the  repeated  inocu- 
lation of  animals  with  cultures  of  this  organism,  killed 
either  by  heat  or  by  chemicals,  it  has  been  found  pos- 
sible to  protect  them  against  otherwise  fatal  doses  of  the 
living  virulent  organism.  When  treated  in  this  way, 
the  goat  supplies  a  serum  that  exhibits  not  only  an 
agglutinating  power  over  the  living  bacilli,  but  pos- 
sesses both  protective  and  curative  properties  when  in- 
jected into  other  susceptible  animals. 

During  1898-1899  Shiga1  employed  a  protective 
serum,  made  after  the  foregoing  principles,  in  the  treat- 
ment of  dysentery  in  human  beings.  During  the  period 
mentioned  he  treated  266  cases,  and  had  a  death-rate  of 

1  See  "  The  Epidemic  Dysentery  of  the  Past  Twenty  Years  in  Japan," 
by  Stuart  Eldridge,  M.  D.,  U.  S.  Marine-Hospital  Service,  Public  Health 
Reports,  1900,  vol.  xv.,  No.  1,  pp.  1-11. 


BACILLUS  DYSENTERIC.  471 

9.(>  j>er  cent. ;  while  for  1736  cases  occurring  at  the  same 
time  and  in  the  same  locality,  but  not  so  treated,  there 
was  a  death-rate  of  34.7  per  cent.1 

Through  the  studies  of  Vedder  and  Duval  the 
observations  of  Shiga,  of  Flexner,  and  of  Kruse,  upon 
acute  dysentery  in  Japan,  in  the  Philippine  Islands,  in 
Puerto  Rico,  and  in  Germany,  are  found  to  be  appli- 
cable to  acute  dysentery  occurring  in  this  country. 
The  micro-organism  described  by  Shiga  was  found  by 
Vedder  and  Duval  in  22  cases  of  acute  dysentery 
occurring  in  Philadelphia,  Lancaster,  Pa.,  and  New 
Haven,  Conn.;  those  in  Lancaster  and  in  New  Haven 
having  been  institutional  outbreaks  of  the  disease.2 

Kruse3  states  that  ^QTOT  gramme  of  dysentery  im- 
mune serum  protects  a  guinea-pig  against  the  minimum 
lethal  dose  of  the  culture.  In  100  cases  treated  with 
the  serum  the  mortality  was  8  per  cent,  as  against  10  to 
11  per  cent,  in  cases  without  serum  treatment. 

Holt4  summarizes  the  results  obtained  in  the  treat- 
ment of  87  cases  with  dysentery  immune  serum.  De- 
cided improvement  was  noted  in  only  12  of  the  patients. 
These  were  principally  hospital  cases,  and  hence  rather 
grave  forms  of  the  disease.  Another  factor  which  prob- 
ably operated  against  the  favorable  influence  of  the 
serum  is  the  fact  that  the  serum  treatment  was  generally 

1  The  foregoing  sketch  is  compiled  from  : 

Shiga:  "  Ueber  deu  Dysenteric-bacillus  (Bacillus  Dyseuterise)," 
Centralblatt  fiir  Bakteriologie  und  Parasitenkunde,  1898,  Abt.  i.  Bd. 
xxiv.  Nos.  22,  23,  24. 

Flexner:  "  On  the  Etiology  of  Tropical  Dysentery,"  Philadelphia 
Medical  Journal,  Sept.  1,  1900. 

2  Journal  Experimental  Medicine,  1902,  vol.  vi.  p.  181. 
8  Kruse :  Deutsche  med.  Wochenschr.,  Jan.  8, 1903. 

4  Holt :  Studies  from  the  Rockefeller  Institute  for  Medical  Research, 
1904,  vol.  ii.  • 


472  BA  CTERWLOG  Y. 

preceded  by  a  careful  bacteriological  analysis  of  the  stools 
in  order  to  establish  a  positive  diagnosis,  requiring  two 
or  three  days  so  that  the  serum  treatment  was  instituted 
late  in  the  course  of  the  disease. 

Holt  points  out  that  the  conditions  necessary  to  obtain 
success  in  the  serum  treatment  of  cases  of  dysentery  are  : 
First,  the  early  use  of  the  serum,  before  serious  lesions 
have  developed  or  before  the  patient's  general  condition 
has  been  too  profoundly  impaired  ;  second,  the  serum 
must  be  administered  in  repeated  doses,  one  or  two  doses 
a  day,  and  continued  for  several  days  in  severe  cases. 


CHAPTER    XXII. 

The  spirillum  (comma  bacillus)  of  Asiatic  cholera — Its  morphological 
and  cultural  peculiarities — Pathogenic  properties — The  bacterio- 
logical diagnosis  of  Asiatic  cholera — Microspira  Metschnikovi — 
Microspira  ("Vibrio")  Schuylkilliensis — Its  morphological,  cul- 
tural, and  pathogenical  characters. 

At  the  conference  held  in  Berlin  in  1884  for  the  pur- 
pose of  discussing  Asiatic  cholera  from  the  sanitary 
aspect,  it  was  announced  by  Koch1  that  he  had  dis- 
covered in  the  intestinal  evacuations  of  individuals  suf- 
fering from  Asiatic  cholera  a  micro-organism  that  he 
believed  to  be  the  cause  of  the  malady.  The  importance 
of  this  statement  necessarily  attracted  widespread  atten- 
tion to  the  subject,  and  as  one  of  the  consequences  there 
existed,  for  a  short  time  following,  some  skepticism  as 
to  the  accuracy  of  Koch's  claim.  These  doubts  arose  as 
a  result  of  a  series  of  contributions  from  other  observers, 
who  endeavored  to  prove  that  the  organism  found  by 
Koch  in  cholera  evacuations  was  common  to  other  local- 
ities, and  was  not  a  specific  accompaniment  of  this  dis- 
ease. It  was  not  very  long,  however,  before  it  was 
evident  that  these  objections  were  based  upon  untrust- 
worthy observations,  and  that  by  reliable  methods  of 
investigation  the  organism,  to  which  he  had  called  atten- 
tion could  be  easily  differentiated  from  each  of  those 
with  which  it  was  claimed  to  be  identical. 

This  organism,  known  both  as  the  spirillum  of  Asiatic 
cholera,  and,  because  of  its  morphology,  as  Koch's 

1  Verhandlungen  der  Conferenz   zur  Erorterung  der  Cholerafrage, 
1884,  Berlin. 

473 


474  BA  CTERIOLOG  Y. 

"  comma  bacillus,"  is  identified  by  the  following  peculi- 
arities : 

MICROSPIRA    COMMA    (KOCH)    SCHROTER,    1886. 

Synonyms :  Comma-bacillus,  Koch,  1884  ;  Spirillum  cholerse  asiatica, 
Fliigge,  1886. 

MORPHOLOGY. — It  is  a  slightly  curved  rod,  ranging 
from  about  0.8  to  2  p.  in  length  and  from  0.3  to  0.4  // 
in  thickness — that  is  to  say,  it  is  usually  from  about  one- 
half  to  two-thirds  the  length  of  the  tubercle  bacillus, 
but  is  thicker  and  plumper.  Its  curve  is  frequently 
not  more  marked  than  that  of  a  comma,  and,  indeed,  it 
is  often  almost  straight ;  at  times,  though,  the  curve  is 
much  more  pronounced,  and  may  even  describe  a  semi- 
circle. Occasionally  the  curve  may  be  double,  one 
comma  joining  another,  with  their  convexities  pointing 
in  opposite  directions,  so  that  a  figure  similar  to  the 
letter  S  is  produced.  In  cultures  long  spiral  or  undu- 
lating threads  may  often  be  seen.  From  these  appear- 
ances this  organism  cannot  be  considered  as  a  bacillus, 
but  rather  as  an  intermediate  type  between  the  bacilli 
and  the  spirilla.  Koch  thinks  it  not  improbable  that 
the  short  comma  forms  represent  segments  of  a  true 
spirillum,  the  normal  form  of  the  organism.  (Fig.  74.) 

It  does  not  form  spores,  and  we  have  no  reliable  evi- 
dence that  it  possesses  the  property  of  entering,  at  any 
time,  a  stage  in  which  its  powers  of  resistance  to  detri- 
mental agencies  are  increased. 

It  is  a  flagellated  organism,  but  has  only  a  single 
flagellum  attached  to  one  of  its  ends. 

It  is  actively  motile,  especially  in  the  comma  stage ; 
though  the  long  spiral  forms  also  possess  this  property. 

GROUPING. — As  found  in  the  slimy  flakes  in  the  intes- 
tinal discharges  from  cholera  patients,  Koch  likens  its 
mode  of  grouping  to  that  seen  in  a  school  of  small  fish 


MICROSPIRA   COMMA.  475 

when  swimming  up  stream  —  i.  e.,  they  all  point  in  nearly 
the  same  direction,  and  lie  in  irregularly  parallel,  linear 


FIG.  74. 


Mierospira  comma.    Impression  cover-slip  from  a  colony  thirty-four 
hours'  old. 

groups  that   are  formed  by  one  comma  being  behind 
the  other  without  being  attached  to  it. 

FIG.  75. 


'•'  *r  '  7*> 
<  <-  **</ 1  ^ 

Involution-forms  of  microspira  comma,  as  seen  in  old  cultures. 

On  cover-slip  preparations  made  from  cultures  in  the 
ordinary  way  there  is  nothing  characteristic  about  the 
grouping ;  but  in  impression  cover-slips  made  from 
young  cultures  the  short  commas  will  nearly  always  be 
seen  in  small  groups  of  three  or  four,  lying  together  in 
such  a  way  as  to  have  their  long  axes  nearly  parallel  to 
one  another.  (See  Fig.  74.) 

In  old  cultures  in  which  development  has  ceased  it 
undergoes  degenerative  changes,  and  the  characteristic 


476  BA  CTER10L  OGY. 

comma  and  spiral  shapes  may  entirely  disappear,  their 
place  being  taken  by  irregular  involution-forms  that 
present  every  variety  of  outline.  (See  Fig.  75.)  In 
this  stage  they  take  on  the  stain  very  feebly,  and  often 
not  at  all. 

CULTURAL  PECULIARITIES. — On  plates  of  nutrient 
gelatin  that  have  been  prepared  from  a  pure  culture  of 
this  organism  and  kept  at  a  temperature  of  from  20° 
to  22°  C.,  development  can  often  be  observed  after  as 
short  a  period  as  twelve  hours,  but  frequently  not  be- 
fore sixteen  to  eighteen  hours.  This  is  especially  true 
of  the  first  or  "  original "  plate,  containing  the  largest 
number  of  colonies.  At  this  time  the  plate  will  pre- 
sent to  the  naked  eye  an  appearance  that  has  been 
likened  to  a  ground-glass  surface,  or  to  a  surface  that 
has  been  stippled  with  a  finely  pointed  needle,  or  one 
upon  which  very  fine  .dust,  lias  been  sprinkled.  This 
appearance  is  due  to  the  presence  of  minute  colonies 
closely  packed  together  upon  the  surface  of  the  gelatin. 
In  the  depth  of  the  gelatin  can  also  be  seen  closely 
packed,  small  points,  likewise  representing  growing 
colonies.  As  growth  progresses  liquefaction  occurs 
around  the  superficial  colonies,  and  in  consequence  this 
plate  is  usually  entirely  liquid  after  from  twenty-four 
to  thirty  hours ;  the  developmental  phases  through  which 
the  colonies  pass  cannot,  therefore,  be  studied  upon  it. 

On  plates  2  and  3,  where  the  colonies  are  more  widely 
separated,  they  can  be  seen  after  twenty-four  to  thirty 
hours  as  small,  round  or  oval,  white  or  cream-white 
points,  and  when  located  superficially  a  narrow  trans- 
parent zone  of  liquefaction  can  be  detected  around 
them.  As  growth  continues  this  liquefaction  extends 
downward  rather  than  laterally,  and  the  colony  ulti- 


MICROSPIRA   COMMA.  477 

mately  assumes  the  appearance  of  a  dense,  white  mass 
lying  at  the  bottom  of  a  sharply-cut  pit  or  funnel  con- 
taining transparent  fluid.  This  liquefaction  is  never 
very  widespread  nor  rapid,  and  rarely  extends  more 
than  one  millimetre  beyond  the  colony  proper.  On 
plates  containing  few  colonies  there  is  little  or  no 
tendency  for  them  to  become  confluent,  and  they  rarely 
exceed  2  to  3  mm.  in  diameter. 

FIG.  76. 


c 
Developmental  phases  of  colonies  of  microspira  comma  at  20°  to  22°  C.  on 

gelatin.    X  about  75  diameters 

a.  After  sixteen  to  eighteen  hours,  b.  After  twenty-four  to  twenty-six 
hours,  c.  After  thirty-eight  to  forty  hours,  d.  After  forty-eight  to  fifty 
hours,  e.  After  sixty-four  to  seventy  hours. 

When  examined  under  a  low  magnifying  lens  the  very 
young  colonies  (sixteen  to  eighteen  hours  old)  appear 
as  pale,  translucent,  granular  globules  of  a  very  delicate 
greenish  or  yellowish-green  color,  sharply  outlined,  and 
not  perfectly  round.  (See  a,  Fig.  76.)  As  growth  pro- 
gresses this  homogeneous  granular  appearance  is  re- 
placed by  an  irregular  tabulation,  and  ultimately  the 
sharply-cut  margin  of  the  colony  becomes  dentated  or 
scalloped.  (See  b  and  c,  Fig.  76.J  After  forty-eight 
hours  (and  frequently  sooner)  liquefaction  of  the  gelatin 


478  BACTERIOLOGY. 

has  taken  place  to  such  an  extent  that  the  appearance 
of  the  colony  is  entirely  altered.  Under  a  magnify- 
ing glass  the  colony  proper  is  now  seen  to  be  ragged 
about  its  edges,  while  here  and  there  shreds  of  the 
colony  can  be  detected  scattered  through  the  liquid 
into  which  it  is  sinking.  These  shreds  evidently  repre- 
sent portions  of  the  colony  that  became  detached  from 
its  margin  as  it  gradually  sank  into  the  liquefied  area. 

At  d,  in  Fig.  76,  is  seen  a  representation  of  the 
several  appearances  afforded  by  the  colonies  at  this 
stage.  At  the  end  of  the  second,  or  during  the  early 
part  of  the  third  day,  the  sinking  of  the  colonies  into 
the  liquefied  pits  resulting  from  their  growth  is  about 
complete,  and  under  a  low-power  lens  they  now  appear  as 
dense,  granular  masses,  surrounded  by  an  area  of  lique- 
faction through  which  can  be  seen  granular  prolonga- 
tions of  the  colony,  usually  extending  irregularly  be- 
tween the  periphery  and  the  central  mass.  (See  ey  Fig. 
76.)  If  the  periphery  be  examined,  it  will  be  seen  to 
be  fringed  with  delicate,  cilia-like  lines  that  radiate 
from  it  in  much  the  same  way  that  cilia  radiate  from 
the  ends  of  the  columnar  epithelial  cells  lining  the  air- 
passages. 

These  are  the  more  marked  phases  through  which  the 
colonies  of  this  organism  pass  in  their  development  on 
gelatin  plates.  In  some  cultures  the  various  phases 
here  given  pass  in  succession  more  quickly,  while  in 
cultures  from  other  sources  they  may  be  somewhat  re- 
tarded. 

On  plates  of  nutrient  agar-agar  the  appearance  of  the 
colonies  is  not  characteristic.  They  appear  as  round  or 
oval  patches  of  growth  that  are  moist  and  moderately 
transparent.  The  colonies  on  this  medium  at  37°  C. 


MICROSPIRA   COMMA. 


479 


naturally  grow  to  a  larger   size  than  do  those  upon 
gelatin  at  22°  C. 

In  stab-cultures  in  gelatin  there  appears  at  the  top 
of  the  needle-track  after  thirty-six  to  forty-eight  hours 
at  22°  C.  a  small,  funnel-shaped  depression.  As  the 
growth  progresses  liquefaction  occurs  about  this  point. 
In  the  centre  of  the  depression  can  be  distinguished 

FIG.  77. 


Stab-culture  of  microspira  comma  in  gelatin,  at  18°  to  20°  C. 

at  18°  to  20°  C» 

a.  After  twenty-four  hours,    b.  After  forty-eight  hours,    c.  After  seventy- 
two  hours,    d.  After  ninety-six  hours. 

a  small,  dense,  whitish  clump,  the  colony  itself.  As 
growth  continues  the  depression  increases  in  extent 
and  ultimately  assumes  an  appearance  that  consists  in 


480  BACTERIOLOGY. 

the  apparent  sinking  of  the  liquefied  portion  in  such 
a  way  as  to  leave  a  perceptible  air-space  between  the 
top  of  the  liquid  and  the  surface  of  the  solid  gelatin. 
The  growth  now  appears  to  be  capped  by  a  small  air- 
bubble.  The  impression  given  by  it  at  this  stage  is  not 
only  that  there  has  been  a  liquefaction,  but  also  a  coin- 
cident evaporation  of  the  fluid  from  the  liquefied  area 
and  a  constriction  of  the  superficial  opening  of  the  fun- 
nel. (See  a,  b}  c,  and  d,  Fig.  77.)  Liquefaction  is  not 
especially  active  along  the  deeper  portions  of  the  track 
made  by  the  needle,  though  in  stab-cultures  in  gelatin  the 
liquefaction  is  much  more  extensive  than  that  usually 
seen  around  colonies  on  plates.  It  spreads  laterally  at 
the  upper  portion,  and  after  about  a  week  a  large  part 
of  the  gelatin  in  the  tube  may  have  become  fluid,  and 
the  growth  will  have  lost  its  characteristic  appearance. 

Stab-  and  smear-cultures  on  agar-agar  present  noth- 
ing characteristic. 

Its  growth  in  bouillon  is  luxuriant,  causing  a  diffuse 
clouding  and  the  ultimate  production  of  a  delicate  film 
upon  the  surface. 

In  sterilized  milk  of  a  neutral  or  amphoteric  reaction 
at  a  temperature  of  36°  to  38°  C.  it  develops  actively, 
and  gradually  produces  an  acid  reaction,  with  coagula- 
tion of  the  casein.  It  retains  its  vitality  under  these 
conditions  for  about  three  weeks  or  more.  The  blue 
color  of  milk  to  which  neutral  litmus  tincture  has  been 
added  is  changed  to  phik  after  thirty-six  or  forty-eight 
hours  at  body-temperature. 

Its  growth  in  peptone  solution,  either  that  of  Dun- 
ham (see  Special  Media)  or  the  one  preferred  by  Koch, 
viz.,  2  parts  of  Witters  peptone,  1  part  of  sodium  chlo^ 
ride,  and  100  parts  of  distilled  water,  is  accompanied  by 


MWROSPIRA   COMMA.  481 

the  production  of  both  indol  and  nitrites,  so  that  after 
eight  to  twelve  hours  in  the  incubator  at  37°  C.  the  rose 
color  characteristic  of  indol  appears  upon  the  addition 
of  sulphuric  acid  alone.  (See  Indol  Reaction.) 

(What  does  the  presence  of  nitrites  in  these  cultures 
signify  ?) 

In  peptone  solution  to  which  rosolic  acid  has  been 
added  the  red  color  is  very  much  intensified  after  four 
or  five  days  at  37°  C. 

Its  growth  on  potato  of  a  slightly  acid  reaction  is  seen 
after  three  or  four  days  at  37°  C.  as  a  dull,  whitish, 
non-glistening  patch  at  and  about  the  site  of  inocula- 
tion. It  is  not  elevated  above  the  surface  of  the  potato, 
and  can  only  be  distinctly  seen  when  held  to  the  light 
in  a  particular  position.  Growth  on  acid  potato  occurs, 
however,  only  at  or  near  the  body-temperature,  owing 
probably  to  the  acid  reaction,  which  is  sufficient  to  pre- 
vent development  at  a  lower  temperature,  but  does  not 
have  this  eifect  when  the  temperature  is  more  favorable. 
On  solidified  blood-serum  growth  is  usually  said  to 
be  accompanied  by  slow  liquefaction.  I  have  not  suc- 
ceeded in  obtaining  this  result  on  Loffler's  serum,  nor 
have  I  detected  anything  characteristic  about  its  growth 
on  this  medium. 

The  temperature  most  favorable  for  its  growth  is 
between  35°  and  38°  C.  It  grows,  but  more  slowly, 
at  17°  C.  Below  16°  C.  no  growth  is  visible. 

It  is  not  destroyed  by  freezing.  When  exposed  to 
65°  C.  its  vitality  is  destroyed  in  five  minutes. 

It  is  strictly  aerobic,  its  development  ceasing  if  the 
supply  of  oxygen  be  cut  off. 

It  does  not  grow  in  an  atmosphere  of  carbonic  acid, 
but  is  not  killed  by  a  temporary  exposure  to  this  gas. 

31 


482  BACTERIOLOGY. 

It  does  not  grow  in  acid  media,  but  flourishes  best  in 
media  of  neutral  or  slightly  alkaline  reaction.  It  is  so 
sensitive  to  the  action  of  acids  that  at  22°  C.  its  devel- 
opment is  arrested  when  an  acid  reaction  equivalent  to 
0.066  to  0.08  per  cent,  of  hydrochloric  or  nitric  acid  is 
present.  (Kitasato.) 

Under  artificial  cultivation  the  maximum  develop- 
ment of  this  organism  is  reached  in  a  comparatively 
short  time ;  after  this  it  remains  quiescent  for  a  period, 
and  finally  degeneration  begins.  The  dying  comma 
bacilli  become  altered  in  appearance  and  assume  the 
condition  known  as  "  involution-forms."  (See  Fig.  75.) 
When  in  this  state  they  take  up  coloring-reagents  very 
faintly  or  not  at  all,  and  may  lose  entirely  their  charac- 
teristic shape. 

When  present  with  other  bacteria,  under  conditions 
favorable  to  growth,  the  comma  bacillus  at  first  grows 
much  more  rapidly  than  do  the  others ;  in  twenty-four 
hours  it  will  often  so  outnumber  the  other  organisms 
present  that  microscopic  examination  might  lead  one 
to  regard  the  material  under  consideration  as  a  pure 
culture  of  this  organism.  Its  conspicuous  develop- 
ment under  these  circumstances  does  not,  however,  last 
longer  than  two  or  three  days ;  degeneration  and  death 
begin,  and  the  other  organisms  gain  the  ascendency. 
This  fact  has  been  taken  advantage  of  by  Schottelius l 
in  the  following  method  devised  by  him  for  the  bac- 
teriological examination  of  dejections  from  cholera 
patients : 

In  dejections  not  examined  immediately  after  being 
passed  it  is  often  difficult,  because  of  the  large  num- 
ber of  other  bacteria  that  may  be  present,  to  detect 

1  Deutsche  med.  Wocheuschrift,  1885,  No.  14. 


MICROSPIRA   COMMA.  483 

with  certainty  the  cholera  organism  by  microscopic 
examination.  It  is  advantageous  in  these  cases  to  mix 
the  dejections  with  about  double  their  volume  of  slightly 
alkaline  beef-tea,  and  allow  them  to  stand  for  about 
twelve  hours  at  a  temperature  between  30°  and  40°  C. 
There  appears  at  the  end  of  this  time,  especially  upon 
the  surface  of  the  fluid,  a  conspicuous  increase  in  the 
number  of  comma  bacilli,  and  cover-slip  preparations 
made  from  the  upper  layers  of  the  fluid  will  reveal  an 
almost  pure  culture  of  this  organism. 

It  is  not  improbable  that  a  similar  process  occurs  in 
the  intestines  of  those  suffering  from  Asiatic  cholera, 
viz.,  a  rapid  multiplication  of  the  comma  bacilli  that 
have  gained  access  to  the  intestines  takes  place,  but  lasts 
for  only  a  short  time,  when  the  comma  bacilli  begin  to 
disappear,  and  after  a  few  days  their  place  is  taken  by 
other  organisms. 

In  connection  with  his  experiments  upon  the  poison 
produced  by  the  cholera  organism  PfeifFer1  states  that 
in  very  young  cultures,  grown  under  access  of  oxy- 
gen, there  is  present  a  body  that  possesses  intensely 
toxic  properties.  This  primary  cholera-poison  stands 
in  very  close  relation  to  the  material  composing  the 
bodies  of  the  bacteria  themselves,  and  is  probably  an 
integral  constituent  of  them,  for  the  vitality  of  the 
cholera  spirilla  can  be  destroyed  by  means  of  chloro- 
form or  thymol,  and  by  drying,  without  apparently 
any  alteration  of  this  poisonous  body.  Absolute  alco- 
hol, concentrated  solutions  of  neutral  salts,  and  a  tem- 
perature of  100°  C.,  decompose  this  substance,  leaving 
intact  secondary  poisons  which  possess  a  similar  physi- 
ological activity,  but  only  when  given  in  from  ten  to 

1  Zeitschrift  fur  Hygiene  uiid  Infektionskrankheiteu,  Bd.  xi.  S.  393. 


484  BACTEEWLOG  Y. 

twenty  times  the  dose  necessary  to  produce  the  same 
effects  with  the  primary  poison. 

EXPERIMENTS  UPON  ANIMALS. — As  a  result  of  ex- 
periments for  the  purpose  of  determining  if  the  disease 
can  be  produced  in  any  of  the  lower  animals  it  has  been 
found  that  white  mice,  monkeys,  cats,  dogs,  poultry,  and 
many  other  animals  are  not  susceptible  to  infection  by  the 
methods  usually  employed  in  inoculation  experiments. 
When  animals  are  fed  on  pure  cultures  of  the  comma 
bacillus  no  effect  is  produced,  and  the  organisms  cannot 
be  obtained  from  the  stomach  or  intestines.  They  are 
destroyed  in  the  stomach,  and  do  not  reach  the  intes- 
tines ;  they  are  not  demonstrable  in  the  faeces  of  these 
animals.  Intravascular  injections  of  a  pure  culture 
into  rabbits  are  followed  by  an  illness,  from  which 
the  animals  usually  recover  in  from  two  to  three  days ; 
intraperitoneal  injections  into  white  mice  are,  as  a  rule, 
followed  by  death  in  from  twenty-four  to  forty-eight 
hours ;  the  conditions  in  both  instances  most  probably 
resulting  from  the  toxic  activities  of  the  poisonous  prod- 
ucts of  growth  of  the  organisms  present  in  the  culture 
employed.  None  of  the  lower  animals  suffer  spontane- 
ously from  Asiatic  cholera. 

The  failure  to  induce  cholera  in  animals  by  feeding, 
or  by  injection  of  cultures  into  the  stomach,  was  shown 
by  Nicati  and  Rietsch1  to  be  due  to  the  destructive 
action  of  the  acid  gastric  juice  on  the  organisms.  They 
showed  that  .if  cultures  of  this  organism  were  intro- 
duced into  the  alimentary  tract  of  certain  animals  in 
such  a  manner  that  they  would  not  be  subjected  to  the 

1  Archiv.  de  Phys.  norm,  et  path.,  1885,  t.  vi.,  3e  ser.   Comptes  rendus, 
xcix.,  p.  928.     Eevue  de  Hygiene,  1885.     Revue  de  Medecine,  1885,  v. 


MICROSPIRA   COMMA.  485 

influence  of  the  gastric  juice,  a  pathological  condition 
closely  simulating  cholera  as  it  occurs  in  man  could  be 
produced.  For  this  purpose  the  common  bile-duct  was 
ligated,  after  which  the  cultures  were  injected  directly 
into  the  duodenum.  Such  interference  with  the  flow 
of  bile  lessens  intestinal  peristalsis,  and  thus  permits 
development  of  the  organisms  at  the  point  at  which 
they  are  deposited — that  is,  the  portion  of  the  intestine 
having  an  alkaline  reaction  and  beyond  the  influence  of 
the  acid  stomach-juice. 

By  this  method  Nicati  and  Rietsch,  Van  Ermengem,1 
Koch,2  and  others  were  enabled  to  produce  in  the  ani- 
mals upon  which  they  operated  a  condition  that  was,  if 
not  identical,  at  all  events  very  similar  pathologically 
to  that  seen  in  the  intestines  of  subjects  dead  of  the 
disease. 

At  a  subsequent  conference  held  in  Berlin  in  1885 
Koch3  described  the  following  method,  by  means  of 
which  he  had  been  able  to  obtain  a  much  greater  de- 
gree of  constancy  in  all  his  eiforts  to  produce  cholera  in 
lower  animals :  bearing  in  mind  the  point  made  by 
Nicati  and  Rietsch  as  to  the  eifect  produced  by  the  acid 
reaction  of  the  gastric  juice,  this  reaction  was  first  to  be 
neutralized  by  injecting  through  a  soft  catheter  passed 
down  the  oesophagus  into  the  stomach  5  c.c.  of  a  5  per 
cent,  solution  of  sodium  carbonate.  Ten  or  fifteen  min- 
utes later  this  was  to  be  followed  by  the  injection  into 
the  stomach  (also  through  a  soft  catheter)  of  10  c.c.  of  a 
bouillon  culture  of  microspira  comma.  For  the  pur- 

1  Recherches  sur  le  Microbe  du  Cholera  Asiatique.  Paris-Bruxelles, 
1H85.     Bull,  de  1'Ar.ad.  roy.  de  Med.  de  Belgique,  xviii.,  3e  ser. 

2  Loc.  cit.  3  Ibid.,  1885. 


486  XA  CTERIOLOG  Y. 

pose  of  arresting  peristalsis  and  permitting  the  bac- 
teria to  remain  in  the  stomach  and  upper  part  of  the 
duodenum  for  as  long  a  time  as  possible,  the  animal  was 
to  receive,  immediately  following  the  injection  of  the 
culture,  an  intraperitoneal  injection,  by  means  of  a 
hypodermic  syringe,  of  1  c.c.  of  tincture  of  opium  for 
each  200  grammes  of  its  body -weight.  Shortly  after 
this  last  injection  deep  narcosis  sets  in  and  lasts  from 
a  half  to  one  hour,  after  which  the  animal  is  as  lively 
as  ever.  Of  35  guinea-pigs  inoculated  in  this  way  by 
Koch,  30  died  of  an  affection  that  was,  in  general,  very 
similar  to  Asiatic  cholera  as  seen  in  man. 

The  condition  of  those  animals  before  death  is  de- 
scribed as  follows  :  twenty-four  hours  after  the  opera- 
tion the  animal  appears  unwell ;  there  is  loss  of  appetite, 
and  the  animal  remains  quiet  in  its  cage.  On  the  fol- 
lowing day  a  paralytic  condition  of  the  hind  extremities 
appears,  which,  as  the  day  wears  on,  becomes  more 
pronounced  ;  the  animal  lies  quite  flat  upon  its  abdomen 
or  on  its  side,  with  legs  extended ;  respiration  is  weak 
and  prolonged,  and  the  pulsations  of  the  heart  are  hardly 
perceptible  ;  the  head  and  extremities  are  cold,  and  the 
body-temperature  is  frequently  subnormal.  The  ani- 
mal usually  dies  after  remaining  in  this  condition  for  a 
few  hours. 

At  autopsy  the  small  intestine  is  found  deeply  in- 
jected and  filled  with  flocculent,  colorless  fluid.  The 
stomach  and  intestines  do  not  contain  solid  masses,  but 
fluid ;  when  diarrhoea  does  not  occur,  firm  scybala  may 
be  detected  in  the  rectum.  Both  by  microscopic  exam- 
ination and  by  culture  methods  the  organisms  are 
found  present  in  the  small  intestine  in  practically  pure 
culture. 


MICROSPIRA   COMMA.  487 

More  recently  Pfeiffer1  has  determined  that  essen- 
tially similar  constitutional  effects  may  be  produced  in 
guinea-pigs  by  the  intraperitoneal  injection  of  rela- 
tively large  numbers  of  this  organism.  His  plan  is  to 
scrape  from  the  surface  of  a  fresh  culture  on  agar-agar 
as  much  of  the  growth  as  can  be  held  upon  a  medium 
size  wire  loop.  This  is  then  finely  divided  in  1  c.c. 
of  bouillon,  and  by  means  of  a  hypodermic  syringe  is 
injected  directly  into  the  peritoneal  cavity.  When  vir- 
ulent cultures  have  been  used  this  operation  is  quickly 
followed  by  a  fall  in  the  temperature  of  the  animal  that 
is  gradual  and  continuous  until  death  ensues,  which  usu- 
ally occurs  in  from  eighteen  to  twenty-four  hours  after 
the  operation,  though  exceptionally  the  animal  recovers, 
even  after  having  exhibited  marked  symptoms  of  pro- 
found toxaemia. 

Continuing  his  studies  upon  this  disease,  Pfeiffer2  has 
demonstrated  that  it  is  possible  to  render  an  animal  tol- 
erant to  or  immune  from  the  poisonous  properties  of  this 
organism  by  repeated  injections  of  non-fatal  doses  of 
dead  cultures  (cultures  that  have  been  killed  by  the 
vapor  of  chloroform  or  by  heat).  He  also  demon- 
strated that  animals  so  immunized  possess  a  specific 
germicidal  action  toward  microspira  comma — i.  e.,  if 
into  the  peritoneal  cavity  of  an  animal  immunized 
from  Asiatic  cholera  living  organisms  be  introduced 
they  will  all  be  destroyed  (disintegrated)  within  a  rela- 
tively short  time.  Furthermore,  if  the  serum  of  an  animal 
immunized  from  cholera  be  injected  into  the  peritoneal 
cavity  of  another  animal  of  the  same  species,  but  not  so 

1  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  Bd.  xi.  and  xiv. 

2  Ibid.,  1894,  Bd.  xvii.  S.  355;  1894,  Bd.  xviii.  S.  1  ;  1895,  Bd.  xx. 
S.  197. 


488  BACTERIOLOGY. 

protected,  and  immediately  afterward  living  cholera  spir- 
illa be  introduced,  a  similar  disintegration  and  destruction 
of  the  bacteria  will  also  result.  He  shows  that  a  more  or 
less  definite  relation  exists  between  the  amount  of 
serum  and  the  number  of  organisms  introduced.  Such 
a  destruction  of  microspira  comma  by  the  serum  of  an 
immunized  animal  does  not  occur  outside  the  animal 
body — that  is,  it  cannot  be  demonstrated  in  a  test-tube, 
unless,  as  Bordet  has  demonstrated,  it  be  perfectly  fresh 
from  the  animal  body,  or,  as  Metschnikoff  has  shown, 
there  be  added  to  it  a  small  quantity  of  fresh  serum 
from  a  normal  guinea-pig.  The  specificity  of  this  reac- 
tion is  suggested  by  Pfeiffer  as  a  means  of  differentiat- 
ing the  cholera  spirillum  from  other  suspicious  species, 
for  no  such  bacteriolytic  action  is  observed  if  species 
other  than  microspira  comma  be  introduced  into  the 
peritoneal  cavity  of  animals  immunized  from  Asiatic 
cholera. 

Pfeiffer  has  further  demonstrated  that  the  serum  of 
animals  artificially  immunized  from  Asiatic  cholera  has 
an  agglutinating  effect  upon  fluid  cultures  of  microspira 
comma  similar  to  that  seen  when  typhoid  bacilli  are 
mixed  with  serum  from  typhoid  cases,  or  from  animals 
artificially  immunized  from  typhoid  infection  or  intoxi- 
cation. (See  Agglutinin.) 

GENERAL  CONSIDERATIONS. — In  all  cases  of  Asiatic 
cholera,  and  only  in  this  disease,  the  organism  just 
described  can  be  detected  in  the  intestinal  evacuations. 
The  more  acute  the  case  and  the  more  promptly  the 
examination  is  made  after  the  evacuations  have  passed 
from  the  patient,  the  less  difficulty  will  be  experienced 
in  detecting  the  organism. 

In  some  cases  the  organism  can  be  detected  in  the 


MICROSPIRA   COMMA.  489 

vomited  matters,  though  by  no  means  so  constantly  as 
in  the  intestinal  contents. 

As  a  rule,  bacteriological  examination  fails  to  reveal 
the  presence  of  the  organisms  in  the  blood  and  internal 
organs  in  this  disease,  though  Nicati  and  Rietsch  claim  to 
have  obtained  them  from  the  common  bile-duct  in  rapidly 
fatal  cases,  and  in  two  out  of  five  cases  they  were  pres- 
ent in  the  gall-bladder.  Doyen  and  Rasstschewsky l 
found  them  in  the  liver  in  pure  culture,  and  Tizzoni 
and  Cattani2  in  both  the  blood  and  the  gall-bladder. 

Microspira  comma  is  a  facultative  saprophyte ;  that 
is  to  say,  it  apparently  finds  in  certain  parts  of  the 
world,  particularly  in  those  countries  in  which  Asiatic 
cholera  is  endemic,  conditions  that  are  not  entirely  un- 
favorable to  its  development  outside  of  the  body.  This 
has  been  found  to  be  the  case  not  only  by  Koch,  who 
detected  the  presence  of  the  organism  in  water-tanks 
in  India,  but  by  many  other  observers  who  have  suc- 
ceeded in  demonstrating  its  growth  under  conditions  not 
embraced  in  the  ordinary  methods  employed  for  the 
cultivation  of  bacteria.3 

The  results  of  experiments  having  for  their  object 
the  determination  of  the  length  of  time  during  which 
this  organism  may  retain  its  vitality  in  water  are  con- 
spicuous for  their  irregularity.  In  the  transactions  of 
the  congress  in  Berlin  for  the  discussion  of  the  cholera 
question,  it  is  stated,  in  connection  with  this  point,  that 
the  experiments  made  with  tank-water  in  India  some- 

1  Reference  to  Vratch,  1885,  in  Allg.  med.  Central  Zeitung,  Berlin. 

2  Centralblatt  fur  die  med.  Wissenschaften,  1886,  No.  43. 

3  Obviously  all  pathogenic  bacteria  that  have  been  isolated  under 
artificial  methods  of  cultivation  are  facultative  saprophytes.  Were  they 
<>f>H(l<ite    parasites,  their    cultivation    upon    dead   materials   would   be 
impossible. 


490  BA  CTERIOLOG  Y. 

times  resulted  in  demonstrating  the  multiplication  of 
the  organisms  introduced  into  it,  while  in  other  cases 
they  died  very  quickly. 

On  February  8, 1884,  microspira  were  found  in  a  tank  at 
Saheb-Began,  in  Calcutta,  and  it  was  possible  to  demon- 
strate them  in  a  living  condition  up  to  February  23d. 

Koch  states  that  in  ordinary  spring-water  or  well- 
water  the  organisms  retained  their  vitality  for  thirty 
days,  whereas  in  the  canal- water  (sewage)  of  Berlin  they 
died  after  six  or  seven  days ;  but  if  this  latter  were 
mixed  with  fsecal  matters,  the  organisms  retained  their 
vitality  for  but  twenty-seven  hours ;  and  in  the  undi- 
luted contents  of  cesspools  it  was  impossible  to  demon- 
strate them  after  twelve  hours.  In  the  experiments  of 
Isicati  and  Rietsch  they  retained  their  vitality  in  steril- 
ized distilled  water  for  twenty  days ;  in  Marseilles  canal- 
water  (sewage),  for  thirty-eight  days;  in  sea-water, 
sixty-four  days ;  in  harbor-water,  eighty-one  days ;  and 
in  bilge-water,  thirty-two  days. 

In  the  experiments  of  Hochstetter,  on  the  other  hand, 
they  died  in  distilled  water  in  less  than  twenty-four 
hours  in  five  of  seven  experiments ;  in  one  of  the  two 
remaining  experiments  they  were  alive  after  a  day,  and 
in  the  other  after  seven  days. 

In  one  test  with  the  water-supply  of  Berlin  the 
organism  retained  its  vitality  for  267  days,  and  in 
another  for  382  days,  notwithstanding  the  fact  that 
many  other  organisms  were  present  at  the  same  time. 
There  is  no  ready  explanation  for  these  variations,  for 
they  depend  apparently  upon  a  number  of  factors  which 
may  act  singly  or  together.  For  example,  in  general  it 
may  be  said  that  the  higher  the  temperature  of  the  water 
in  which  these  organisms  are  present,  up  to  20°  C.,  the 


MICEOSPIRA   COMMA.  491 

longer  do  they  retain  their  vitality  ;  the  purer  the  water — 
that  is,  the  poorer  in  organic  matters — the  more  quickly 
do  the  organisms  die,  whereas  the  richer  it  is  in  organic 
matter  the  longer  do  they  retain  their  vitality. 

Still  another  point  that  must  be  considered  in  this 
connection  is  the  antagonistic  influences  under  which 
they  find  themselves  when  placed  in  water  containing 
large  numbers  of  organisms  that  are,  so  to  speak,  at 
home  in  water — the  so-called  normal  water-saprophytes. 

The  effect  of  light  upon  growing  bacteria  must  not 
be  lost  sight  of,  for  it  has  been  shown  that  a  surprisingly 
large  number  of  these  organisms  are  robbed  of  their 
vitality  by  a  relatively  short  exposure  to  the  rays  of 
the  sun ;  and  it  is  therefore  not  unlikely  that  the  non- 
observance  of  this  fact  may  be,  in  part  at  least,  account- 
able for  some  of  the  discrepancies  that  appear  in  the 
results  of  these  experiments. 

In  his  studies  upon  the  behavior  of  pathogenic  and 
other  micro-organisms  in  the  soil  Carl  Frankel1  found 
that  microspira  comma  was  not  markedly  susceptible 
to  those  deleterious  influences  that  cause  the  death  of 
a  number  of  other  pathogenic  organisms.  During  the 
months  of  August,  September,  and  October  cultures  of 
the  comma  bacillus  that  had  been  buried  in  the  ground 
at  a  depth  of  three  metres  retained  their  vitality ;  on 
the  other  hand,  in  other  months,  particularly  from 
April  to  July,  they  lost  their  vitality  when  buried  to 
the  depth  of  only  two  metres.  At  a  depth  of  one  and 
a  half  metres  vitality  was  not  destroyed,  and  there  was 
a  regular  development  in  cultures  so  placed. 

As  a  result  of  experiments  performed  in  the  Imperial 
Health  Bureau  at  Berlin,  it  was  found  that  the  bodies  of 

1  Zeitschrift  fur  Hygiene,  Bd.  ii.  S.  521. 


492  BACTERIOLOGY. 

guinea-pigs  that  had  died  of  cholera  induced  by  Koch's 
method  of  inoculation  contained  no  living  cholera  spir- 
illa when  exhumed  after  having  been  buried  for  nineteen 
days  in  wooden  boxes,  or  for  twelve  days  in  zinc  boxes. 
In  a  few  that  had  been  buried  in  moist  earth,  without 
having  been  encased  in  boxes,  when  exhumed  after  two 
or  three  months,  the  results  of  examinations  for  cholera 
spirilla  were  likewise  negative. 

Kitasato,1  in  his  experiments  with  the  cholera  organ- 
ism, found  that  when  mixed  with  the  intestinal  evacu- 
ations of  human  beings  under  ordinary  conditions  it 
lost  its  vitality  in  from  a  day  and  a  half  to  three 
days.  If  the  evacuations  were  sterilized  before  the 
cultures  were  mixed  with  them,  the  organisms  retained 
their  vitality  from  twenty  to  twenty-five  days.  He 
was  unable  to  come  to  any  definite  conclusion  as  to  the 
cause  of  these  phenomena. 

It  was  demonstrated  by  Hesse 2  and  by  Celli 3  that 
many  substances  commonly  employed  as  food-stuffs 
serve  as  favorable  materials  for  the  development  of 
the  cholera  organisms.  In  his  experiments  upon  its 
behavior  in  milk  Kitasato 4  found  that  at  a  temperature 
of  36°  C.  microspira  comma  developed  very  rapidly 
during  the  first  three  or  four  hours,  and  outnumbered 
the  other  organisms  commonly  found  in  milk.  They 
then  diminished  in  number  from  hour  to  hour  as  the 
acidity  of  the  milk  increased,  until  finally  their  vitality 
was  lost;  at  the  same  time  the  common  saprophytic 
bacteria  increased  in  number.  Relatively  the  same 
process  occurs  at  a  lower  temperature,  from  22°  to  25° 

1  Zeitschrift  fur  Hygiene,  Bd.  v.  S.  487. 

2  Ibid.,  Bd.  v.  S.  527. 

3  Bolletino  della  R.  Acad.  Med.  di  Roma,  1888. 

4  Zeitschrift  fur  Hygiene,  Bd.  v.  S.  491. 


MICROSPIRA   COMMA.  493 

C. ;  but  it  is  slower,  the  maximum  development  of  the 
cholera  organisms  being  reached  at  about  the  fifteenth 
hour,  after  which  time  they  were  outnumbered  by  the 
ordinary  saprophytes  present. 

From  the  foregoing  it  would  seem  that  the  vitality  of 
microspira  comma  in  milk  depends  largely  upon  the 
reaction ;  the  more  quickly  the  milk  becomes  sour  the 
more  quickly  does  the  organism  become  inert ;  while  the 
longer  the  milk  retains  its  neutral,  or  only  very  slightly 
acid,  reaction,  the  longer  do  the  cholera  organisms  that 
may  be  present  in  it  retain  their  power  of  multiplication. 

According  to  Laser,1  the  cholera  organism  retains  its 
vitality  in  butter  for  about  seven  days ;  it  is  therefore  pos- 
sible for  the  disease  to  be  contracted  by  the  use  of  butter 
that  has  in  any  way  been  in  contact  with  cholera  material. 

In  regard  to  the  antagonism  between  the  microspira 
comma  and  other  organisms  with  which  it  may  come 
in  contact,  the  experiments  of  Kitasato2  led  him  to 
conclude  that  no  organism  has  been  found  which, 
when  growing  in  the  same  culture-medium  with  it, 
possessed  the  power  of  depriving  it  of  vitality  within 
a  short  time.  On  the  other  hand,  the  experiments 
showed  that  there  were  quite  a  number  of  other  organ- 
isms the  development  of  which  was  checked,  and  in 
some  cases  their  vitality  was  completely  destroyed,  when 
growing  in  the  same  medium,  with  the  microspira  comma. 

From  this  it  woujd  appear  that  the  disappearance  of 
the  microspira  comma  from  mixed  cultures  and  from 
the  evacuations  in  the  short  time  mentioned  is  due  more 
to  unfavorable  nutritive  conditions  than  to  the  direct 
action  of  the  other  organisms  present. 

1  Laser  :  Zeitschrift  fur  Hygiene,  Bd.  x.  S.  513. 
••'Kitasato:  Ibid.,  Bd.  vi.  S.  1. 


494  BACTERIOLOGY. 

When  completely  dried,  according  to  Koch,  micro- 
spira  comma  does  not  retain  its  vitality  longer 
than  twenty-four  hours,  but  by  others  its  vitality  is 
said  to  be  destroyed  by  an  absolute  drying  of  three 
hours.  In  moist  conditions,  as  in  artificial  cultures, 
vitality  may  be  retained  for  many  months ;  though  re- 
peated observations  lead  us  to  believe  that  under  these 
circumstances  virulence  is  diminished.  According  to 
Kitasato,1  they  retain  their  vitality  when  smeared  upon 
thin  glass  cover-slips  and  kept  in  the  moist  chamber 
for  from  85  to  100  days,  and  for  as  long  as  200  days 
when  deposited  upon  bits  of  silk  thread. 

In  the  course  of  his  studies  upon  the  persistency  of 
pathogenic  micro-organisms  in  the  dead  body  von 
Esmarch 2  found  that  when  the  cadaver  of  a  guinea- 
pig  dead  after  the  introduction  of  cholera  organisms 
into  the  stomach  was  immersed  in  water  until  decom- 
position set  in,  after  eleven  days,  when  decomposition 
was  far  advanced,  it  was  impossible  to  find  any  living 
microspira  comma  by  the  ordinary  plate  methods. 

A  similar  experiment  resulted  in  their  disappearance 
in  five  days.  In  another  experiment,  in  which  de- 
composition was  allowed  to  go  on  without  the  animal 
being  immersed  in  water,  none  could  be  detected  after 
the  fifth  day. 

Carl  Frankel 3  has  shown  that  an  atmosphere  of  car- 
bonic acid  is  directly  inhibitory  to  the  development  of 
microspira  comma,  and  Percy  Frankland 4  states  that 
in  an  atmosphere  of  this  gas  it  dies  in  about  eight  days. 

1  Kitasato :  Zeitschrift  fur  Hygiene,  Bd.  v.  S.  134. 

2  v.  Esmarch :  Ibid.,  Bd.  vii.  S.  1. 

s  Carl  Frankel :  Ibid.,  Bd.  v.  S.  332. 
*  Percy  Frankland:  Ibid.,  Bd.  vi.  S.  13. 


THE  DIAGNOSIS  OF  ASIATIC  CHOLERA.       495 

In  an  atmosphere  of  carbon  monoxide  its  vitality  is  lost 
in  nine  days,  and  in  general  the  same  may  be  said  for  it 
when  exposed  to  an  atmosphere  of  nitrous  oxide  gas. 

From  what  has  been  said,  we  see  that  the  spirillum 
of  Asiatic  cholera,  while  possessing  the  power  of  pro- 
ducing in  human  beings  one  of  the  most  rapidly  fatal 
diseases  with  which  we  are  acquainted,  is  still  one  of  the 
least  resistant  of  the  pathogenic  organisms  known  to  us. 
Under  conditions  most  favorable  to  its  growth  its  de- 
velopment is  self-limited  ;  it  is  markedly  susceptible  to 
acids,  alkalies,  other  chemical  disinfectants,  and  heat ; 
but  when  partly  dried  upon  clothing,  food,  or  other 
objects,  it  may  retain  its  vitality  for  a  relatively  long 
period  of  time,  and  it  is  more  than  probable  that  in  this 
way  the  disease  is  often  disseminated  from  points  in 
which  it  is  epidemic  or  endemic  into  localities  that  are 
free  from  it. 

THE    DIAGNOSIS    OF    ASIATIC    CHOLERA    BY    BACTERIO- 
LOGICAL   METHODS. 

Because  of  the  manifold  channels  that  are  open  for 
the  dissemination  of  this  disease  it  is  of  the  utmost 
importance  that  it  should  be  recognized  as  quickly 
as  possible,  for  with  every  moment  of  delay  in  its 
recognition  opportunities  for  its  spread  multiply.  It 
is  essential,  therefore,  when  employing  bacteriological 
means  for  making  the  diagnosis,  to  bear  in  mind  those 
biological  and  morphological  features  of  the  organism 
that  appear  most  quickly  under  artificial  methods  of 
cultivation,  and  which,  at  the  same  time,  may  be  con- 
sidered as  characteristic  of  it,  viz.,  its  peculiar  mor- 
phology and  grouping  ;  the  much  greater  rapidity  of  its 
growth  over  that  of  other  bacteria  with  which  it  may 


496  BACTERIOLOGY. 

be  associated ;  the  characteristic  appearance  of  its  col- 
onies on  gelatin  plates  and  of  its  growth  in  stab-cultures 
in  gelatin  ;  its  property  of  producing  indol  and  coinci- 
dently  nitrites  in  from  six  to  eight  hours  in  peptone 
solution  at  37°  to  38°  C. ;  and  its  power  of  causing  the 
death  of  guinea-pigs  in  from  sixteen  to  twenty-four 
hours  when  introduced  into  the  peritoneal  cavity,  death 
being  preceded  by  symptoms  of  extreme  toxaemia,  char- 
acterized by  prostration  and  gradual  and  continuous 
fall  in  the  temperature  of  the  animal's  body. 

In  1893  Koch1  called  attention  to  a  plan  of  pro- 
cedure that  comprehends  the  points  just  enumerated. 
By  its  employment  the  diagnosis  can  be  established  in 
the  majority  of  cases  of  Asiatic  cholera  in  from  eighteen 
to  twenty-two  hours.  In  general,  the  steps  to  be  taken 
and  points  to  be  borne  in  mind  are  as  follows :  the 
evacuations  should  be  examined  as  soon  as  possible 
after  they  have  been  passed. 

MICROSCOPIC  EXAMINATION. — 1.  From  one  of  the 
small  slimy  particles  seen  in  the  semi-fluid  evacuations 
prepare  a  cover-slip  preparation  in  the  ordinary  way 
and  stain  it.  If,  upon  microscopic  examination,  only 
curved  rods,  or  curved  rods  greatly  in  excess  of  all 
other  forms,  are  present,  the  diagnosis  of  Asiatic  cholera 
is  more  than  likely  correct ;  and  particularly  is  this  true 
if  these  organisms  are  arranged  in  irregular  linear 
groups  with  the  long  axes  of  all  the  rods  pointing  in 
nearly  the  same  direction — that  is  to  say,  somewhat  as 
minnows  arrange  themselves  when  swimming  up  stream 
in  schools.  (Koch.) 

In  1886  Weisser  and  Frank2  expressed  their  opinion 

1  Zeitschrift  fur  Hygiene  und  Itifiktionskleiten,  1893,  Bd.  xiv.  S.  319. 

2  Ibid.,  1886,  Bd.  i.  S.  379. 


THE  DIAGNOSIS  OF  ASIATIC  CHOLERA.       497 

upon  the  value  of  microscopic  examination  in  these  cases 
as  follows : 

a.  In  the  majority  of  cases  microscopic  examination 
is  sufficient  for  the  detection  of  the  presence  of  micro- 
spira  comma  in  the   intestinal   evacuations  of  cholera 
patients. 

b.  Even  in  the  most  acute  cases,  running  a  very  rapid 
course,  microspira  comma  can  always  be  found  in  the 
evacuations. 

c.  In  general,  the  number  of  microspira  comma  pres- 
ent is  greater  the  earlier  death  occurs  ;  when  death  is 
delayed,  and  the  disease  continues  for  a  long  period, 
their  number  is  diminished. 

(1.  Should  the  patient  not  die  of  cholera,  but  of 
some  other  disease,  such  as  typhoid  fever,  that  may  be 
engrafted  upon  it,  microspira  comma  may  disappear 
entirely  from  the  intestines. 

2.  From  another  slimy  flake  prepare  a  set  of  gelatin 
plates.     Expose  them  to  a  temperature  of  from  20°  to 
22°  C.,  and  after  sixteen,  twenty-two,  and    thirty-six 
hours  observe  the  appearance  of  the  colonies.     Usually 
after  about  twenty-two  hours  the  colonies  of  this  organ- 
ism can  easily  be  identified  by  one  familiar  with  them. 

3.  With  another  slimy  flake  start  a  culture  in  a  tube 
of  peptone  solution — either  the    solution   of   Dunham 
or,  as  Koch  proposes,  a  solution  of  double  the  strength 
of  that  of  Dunham  (Witte's  peptone  is  to  be  used,  as  it 
gives  the  best  and  most  constant  results).     Keep  this  at 
from  37°  to  38°  C.,  and  at  the  end  of  from  six  to  eight 
hours  prepare  cover-slips  from  the  upper  layers  (without 
shaking)  and  examine  them  microscopically.    If  comma 
bacilli  were  present  in  the  original  material,  and  are 
capable  of  multiplication,  they  will  be  found  in  this  local- 

32 


498  BA  CTERIOLOG  Y. 

ity  in  almost  pure  culture.  After  the  microscopic  exami- 
nation prepare  a  second  peptone  culture  from  the  upper 
layers  of  the  one  just  examined,  also  a  set  of  gelatin 
plates,  and  with  what  remains  make  the  test  for  indol 
by  the  addition  of  10  drops  of  concentrated  sulphuric; 
acid  for  each  10  c.c.  of  fluid  contained  in  the  tube.  If 
comma  bacilli  are  growing  in  the  tube,  the  rose  color 
characteristic  of  the  presence  of  indol  should  appear. 

By  following  this  plan  "  a  bacteriologist  who  is 
familiar  with  the  morphological  and  biological  peculi- 
arities of  this  organism  should  make  a  more  than  prob- 
able diagnosis  at  once  by  microscopic  examination  alone, 
and  a  positive  diagnosis  in  from  twenty  to,  at  most, 
twenty-four  hours  after  beginning  the  examination." 
(Koch.) 

In  certain  doubtful  cases  the  organisms  are  present  in 
the  intestinal  canal  in  very  small  numbers,  and  micro- 
scopic examination  is  not,  therefore,  of  so  much  assist- 
ance. In  these  cases  plates  of  agar-agar,  of  gelatin, 
and  cultures  in  the  peptone  solution  should  be  made. 

The  plates  of  agar-agar  should  not  be  prepared  in 
the  usual  way,  but  the  agar-agar  should  be  poured  into 
Petri  dishes  and  allowed  to  solidify,  after  which  one  of 
the  slimy  particles  may  be  smeared  over  its  surface. 
The  comma  bacillus,  being  markedly  aerobic,  develops 
very  much  more  readily  when  its  colonies  are  located 
upon  the  surface  then  when  in  the  depths  of  the  med- 
ium. A  point  to  which  Koch  calls  attention,  in  con- 
nection with  this  step  in  manipulation,  is  the  necessity 
of  having  the  surface  of  the  agar-agar  free  from 
the  water  squeezed  from  it  when  it  solidifies,  as  the. 
presence  of  the  water  interferes  with  the  development 
of  the  colonies  at  isolated  points  and  causes  them  to 


THE  DIAGNOSIS  OF  ASIATIC  CHOLERA.       499 

become  confluent.  To  obviate  this  he  recommends  that 
the  agar-agar  be  poured  into  the  plates  and  the  water 
allowed  to  separate  from  the  surface  at  the  temperature 
of  the  incubator  before  they  are  used.  It  is  wise,  there- 
fore, when  one  is  liable  to  be  called  on  for  such  work 
as  this,  to  keep  a  number  of  sterilized  plates  of  agar- 
agar  in  the  incubator  ready  for  use,  just  as  sterilized 
tubes  of  the  media  are  always  ready  at  hand.  The 
advantage  of  using  the  agar  plates  is  the  higher  tem- 
perature at  which  they  can  be  kept,  and  consequently 
a  more  favorable  condition  for  the  development  of  the 
colonies. 

As  soon  as  isolated  colonies  appear  they  should 
be  examined  microscopically  for  the  presence  of  bac- 
teria having  the  morphology  of  the  one  we  are  seek- 
ing, and  as  soon  as  such  is  detected  gelatin  plates 
and  cultures  in  peptone  solution  (for  the  indol  reac- 
tion) should  be  made.  The  peptone  culture  from  the 
original  material  should  be  examined  microscopically 
from  hour  to  hour  after  the  sixth  hour  that  it  has 
been  in  the  incubator.  The  material  taken  for  ex- 
amination should  always  come  from  near  the  surface 
of  the  fluid,  and  care  should  be  taken  not  to  shake 
the  tube.  As  soon  as  comma  bacilli  are  detected  in 
considerable  numbers  in  the  upper  layers  of  the 
fluid  agar-agar  plates  and  fresh  peptone  cultures 
should  be  made  from  them.  In  from  ten  to  twelve 
hours  at  37°  C.  the  colonies  will  develop  on  the  agar- 
agar  plates  to  a  size  sufficient  for  recognition  oy  micro- 
scopic examination,  and  from  this  examination  an 
opinion  can  usually  be  formed.  This  opinion  should 
always  be  controlled  by  cultures  in  the  peptone  solution 
made  from  each  of  several  single  colonies,  and  finally 


500  BACTERIOLOG  Y. 

the  test  for  the  presence  or  absence  of  indol  in  these 
cultures  should  be  made. 

In  all  doubtful  cases,  in  which  only  a  few  curved 
bacilli  are  present,  or  in  which  irregularities  in  either 
the  rate  or  mode  of  their  development  occur,  pure  cult- 
ures should  be  obtained  as  soon  as  possible  by  the  agar- 
agar  plate  method  and  by  the  method  of  cultivation  in 
peptone  solution,  and  their  virulence  tested  upon  ani- 
mals. For  this  purpose  cultures  upon  agar-agar  from 
single  colonies  must  be  made.  From  the  surface  of 
one  of  such  cultures  a  large  wire-loopful  should  be 
scraped  and  broken  up  in  about  one  cubic  centimetre 
of  bouillon,  and  the  suspension  thus  made  injected 
by  means  of  a  hypodermic  syringe  directly  into  the 
peritoneal  cavity  of  a  guinea-pig  of  about  350  to 
400  grammes  weight.  For  larger  animals  more  mate- 
rial is  used.  If  the  material  injected  is  from  a 
fresh  culture  of  the  cholera  organism,  toxic  symp- 
toms at  once  appear;  these  have  their  most  pro- 
nounced expression  in  depression  of  temperature,  and 
if  one  follows  this  decline  in  temperature  from  time  to 
time  with  the  thermometer  it  will  be  seen  to  be  gradual 
and  continuous  from  the  time  of  injection  to  the  death 
of  the  animal  (Pfeiffer1),  which  occurs  in  from  eighteen 
to  twenty-four  hours  after  the  operation. 

In  general,  this  is  the  procedure  employed  in  the  In- 
stitute for  Infectious  Diseases  at  Berlin,  under  .Koch's 
direction. 

1  Loc.  cit. 


MICROSPIRA   METCHNIKOVL  501 

MICROSPIRA  METCHNIKOVI  (GAMALEIA),  MIGULA, 
1900. 

Synonym :  Vibrio  Metchnikovi,  Gamaleia,  1888. 

A  spirillum  that  simulates  very  closely  the  comma 
bacillus  of  cholera  in  its  morphological  and  cultural 
peculiarities,  but  which  is  still  easily  distinguished  from 
it,  is  that  described  by  Gamaleia l  under  the  name  of 

FIG.  78. 


Microspira  Metchnikovi  from  agar-agar  culture,  twenty-four  hours'  old. 

microspira  Metchnikovi.  It  was  found  post  mortem  in  a 
number  of  fowls  that  had  died  in  the  poultry-market  of 
Odessa,  and  the  experiments  of  the  discoverer  led  him 
to  believe  that  it  was  related  etiologically  to  the  gastro- 
enteritis from  which  the  chickens  had  been  suffering. 

Morphologically  it  appears  as  short,  curved  rods  and  as 
longer,  spiral-like  filaments.  It  is  usually  thicker  than 
Koch's  spirillum  and  is  at  times  much  longer,  while 
again  it  is  seen  to  be  shorter.  It  is  usually  more  dis- 
tinctly curved  than  the  "  comma  bacillus."  (Fig.  78.) 

It  is  supplied  with  a  single  flagellum  at  one  of  its 
extremities,  and  is  therefore  motile. 

It  does  not  form  spores. 

It  is  aerobic. 

Its  growth  upon  gelatin  plates  is  usually  character- 

1  Annales  de  1'Institut  Pasteur,  1888,  tome  ii.,  pp.  482,  552. 


502  BACTERIOLOGY. 

ized,  according  to  Pfeiffer,  by  the  appearance  of  two 
kinds  of  liquefying  colonies,  one  strikingly  like  those 
of  the  Finkler-Prior  organism,  the  other  very  similar 
to  those  produced  by  Koch's  comma  bacillus,  though  in 
both  cases  the  liquefaction  resulting  from  the  growth  of 
this  organism  is  more  energetic  than  that  common  to 
the  spirillum  of  Asiatic  cholera.  After  from  twenty- 
four  to  thirty  hours  the  medium-size  colonies,  when 
examined  under  a  low  power  of  the  microscope,  show  a 
yellowish-brown,  ragged  central  mass  surrounded  by  a 
zone  of  liquefaction  that  is  marked  by  a  border  of  deli- 
cate radii.  (Fig.  79.) 


Colony  of  microspira  Metchnikovi  in  gelatin,  after  thirty  hours  at  20°  to 
22°  C.     X  about  75  diameters. 

In  gelatin  stab-cultures  the  growth  has  much  the 
same  general  appearance  as  that  of  the  cholera  spiril- 
lum, but  is  exaggerated  in  degree.  The  liquefaction 
is  far  more  rapid,  and  the  characteristic  appearance 
of  the  growth  is  lost  in  from  three  to  four  days. 
(See  a,  6,  c,  d,  Fig.  89.)  Development  and  liquefaction 
along  the  deeper  parts  of  the  needle-track  are  much 
more  pronounced  than  is  the  case  with  the  "  comma 
bacillus." 

Its  growth  on  agar-agar  is  rapid ;  after  twenty-four 
to  forty-eight  hours  a  grayish  deposit  appears,  which  has 
a  tendency  to  become  yellowish  with  age. 

On  potato  at  37°  C.  its  growth  is  seen  as  a  moist, 
coffee-colored  patch,  surrounded  by  a  much  paler  zone. 


MICROSPIRA  METCHNIKOVI. 


503 


The  whole  growth  is  so  smooth  and  glistening  that  it 
has  somewhat  the  appearance  of  being  varnished. 

In  bouillon  it  quickly  causes  opacity,  with  the  ulti- 
mate production  of  a  delicate  pellicle  upon  the  surface. 


FIG.  80. 


a  b  c  d 

Stab-culture  of  microspira  Metchnikovi  in  gelatin,  at  18°  to  20°  C. 

a.  After  twenty-four  hours,    b.  After  forty-eight  hours,   c.  After  seventy-two 
hours,    d.  After  ninety-six  hours. 


It  causes  liquefaction  of  blood-serum,  the  liquefied 
area  being  covered  by  a  dense,  wrinkled  pellicle. 

When  grown  in  peptone  solution  it  produces  indol 
and  coincidently  nitrites,  so  that  the  rose-colored  reac- 
tion characteristic  of  indol  is  obtained  by  the  addi- 


504  BA  CTERIOLOG  Y. 

tion  of  sulphuric  acid  alone.  The  production  of  indol 
by  this  organism  is  usually  greater  than  that  com- 
mon to  the  comma  bacillus  under  the  same  circum- 
stances. 

In  milk  it  causes  an  acid  reaction  with  coagulation  of 
the  casein.  The  coagulated  casein  collects  at  the  bot- 
tom of  the  tube  in  irregular  masses,  above  which  is  a 
layer  of  clear  whey.  If  blue  litmus  has  been  added 
to  the  milk,  the  color  is  changed  to  pink  in  from 
twenty-four  to  thirty  hours,  and  after  forty-eight  hours 
decolorization  and  coagulation  occur.  The  clots  of 
casein  are  not  re-dissolved.  After  about  a  week  the 
acidity  of  the  milk  is  at  its  maximum,  and  the  organ- 
isms quickly  die. 

It  causes  the  red  color  of  the  rosolic-acid-peptone 
solution  to  become  very  much  deeper  after  four  or  five 
days  at  37°  C. 

It  does  not  cause  fermentation  of  glucose  with  pro- 
duction of  gas. 

It  is  killed  in  five  minutes  by  a  temperature  of  50°  C. 
(Stern  berg.) 

It  is  pathogenic  for  chickens,  pigeons,  and  guinea- 
pigs.  Rabbits  and  mice  are  affected  only  by  very  large 
doses. 

Gamaleia  states  that  chickens  affected  with  the  chol- 
eraic gastro-enteritis  of  which  this  organism  is  the  cause, 
are  usually  seen  sitting  quietly  with  ruffled  feathers. 
They  suffer  from  diarrhea,  but  there  is  no  elevation 
of  temperature.  Hyperaemia  of  the  entire  gastro-intes- 
tinal  tract  is  seen  at  autopsy.  The  other  internal  organs 
do  not,  as  a  rule,  present  anything  abnormal  to  the 
naked  eye.  The  intestinal  canal  contains  yellowish 
fluid  with  which  blood  may  be  mixed.  In  adult  chickens 


MICROSPIRA   METCHNIKOVL  505 

the  spirilla  are  not  found  in  the  blood,  but  in  young 
ones  they  are  usually  present  in  small  numbers. 

After  the  introduction  into  the  pectoral  muscle  of  a 
very  small  quantity  of  a  culture  of  this  organism 
pigeons  succumb  in  from  eight  to  twenty  hours.  The 
most  conspicuous  post-mortem  lesion  is  found  at  the  site 
of  inoculation.  The  muscle  is  marked  by  yellow, 
necrotic  stripes ;  is  more  or  less  redematous ;  is  swollen, 
and  contains  the  vibrios  in  enormous  numbers.  The 
intestines  are  usually  filled  with  fluid  contents,  which 
may  or  may  not  be  blood-stained  ;  the  walls  of  the  in- 
testines are  often  injected  with  blood,  and  occasionally 
markedly  so.  The  conditions  of  the  other  internal 
viscera  are  inconstant.  In  fatal  cases  the  vibrios  are 
present  in  large  numbers  in  the  blood  and  internal 
organs.  In  pigeons  that  survive  inoculation  the  organ- 
isms may  be  found  only  at  the  site  of  inoculation,  or 
I  very  sparingly  in  the  blood  also.  These  animals  usually 
exhibit  immunity  from  subsequent  inoculations.  In 
certain  instances  the  results  of  infection  are  chronic; 
the  inoculated  pectoral  muscle  atrophies,  the  pigeon  loses 
in  weight  and  finally  dies  after  one  or  two  weeks.  In 
these  cases  the  organisms  are  usually  absent  from  the 
blood  and  internal  organs,  and  may  even  be  absent  from 
the  site  of  inoculation,  or,  if 'present,  in  only  very  small 
number. 

Guinea-pigs  usually  die  in  from  twenty  to  twenty- 
four  hours  after  subcutaneous  inoculation.  At  autopsy 
an  extensive  oedema  of  the  subcutaneous  tissues  about 
the  seat  of  inoculation  is  seen,  and  there  is  usually 
a  necrotic  condition  of  the  tissues  in  the  vicinity  of  the 
point  of  puncture.  As  the  blood  and  internal  organs 
of  both  pigeons  and  guinea-pigs  contain  the  vibrios  in 


50G  BACTERIOLOGY. 

large  numbers,  the  infection  in  these  animals  takes, 
therefore,  the  form  of  acute,  general  septicaemia. 

The  blood-serum  of  both  pigeons  and  guinea-pigs 
that  have  survived  inoculation  with  this  organism — i.  e., 
that  have  acquired  immunity  from  it — is  bactericidal 
in  vitro  for  this  organism.  It  also  possesses  a  certain 
degree  of  immunity-conferring  property,  as  may  be 
demonstrated  by  injecting  it  into  normal  pigeons  and 
guinea-pigs  that  are  subsequently  to  be  inoculated  with 
virulent  cultures. 

Very  old  cultures  of  this  organism  in  bouillon  be- 
come distinctly  alkaline  in  reaction.  At  this  stage  they 
contain  a  toxin  that  is  markedly  active  for  susceptible 
animals.  This  toxin  is  not  dissolved  in  the  fluid  to  any 
extent,  but  is  apparently  in  intimate  association  with  the 
proteid  matters  composing  the  bacteria. 

Gastro-enteritis  may  be  produced  in  both  chickens 
and  guinea-pigs  by  feeding  them  with  food  with  which 
cultures  of  this  organism  have  been  mixed.  (Gamaleia.) 

MICROSPIRA  SCHUYLKILLIENSIS,  ABBOTT,  1896. 
Synonym  :  Vibrio  Schuylkilliensis,  Abbott,  1896. 

Abbott  reports  the  discovery  of  a  microspira  in  the 
water  of  the  Schuylkill  River,  at  Philadelphia,  and  later, 
Bergey1  reports  the  presence  of  the  same  organism, 
as  well  as  several  varieties  that  are  slightly  different, 
in  the  waters  of  the  Schuylkill  and  Delaware  rivers, 
along  the  entire  city  front,  more  especially  in  the  efflu- 
ents of  the  sewers. 

Microspira  Schuylkilliensis  is  a  short,  rather  plump 
"  comma,"  often  with  a  very  decided  curve,  with 
rounded  or  slightly  pointed  ends.  As  usually  seen  it 

1  Bergey:  Jour,  of  Exper.  Mod.,  vol.  ii.,  1897,  p.  535. 


MICROSPIRA  SCHUYLKILL1ENSIS.  507 

is  a  little  shorter  and  thicker  than  the  microspira 
comma,  though  this  feature  is  quite  variable.  It  is 
actively  motile,  having  a  single  polar  flagellum.  It 
does  not  form  spores.  It  stains  with  the  ordinary  ani- 
line stains,  but  is  negative  to  Gram's  method. 

COLONIES  ON  GELATIN. — The  colonies  are  sharply 
defined,  distinctly  granular,  and  have  usually  fine 
irregular  markings,  as  if  they  were  creased  or  folded. 
Sometimes  they  present  indistinct  concentric  mark- 
ings. As  growth  progresses  these  markings  be- 
come more  and  more  distinct  and  finally  give  to 
the  colony  a  decidedly  lobulated  or  mulberry-like 
appearance. 

After  about  the  third  or  fourth  day,  when  liquefac- 
tion is  actively  in  progress,  the  majority  of  the  colonies 
lose  their  characteristic  appearance.  They  are  seen  as 
irregular,  ragged,  granular  masses  lying  in  the  centre 
of  pits  of  liquefied  gelatin. 

GELATIN  STAB  CULTURES. — In  stab  cultures  in  gel- 
atin the  appearance  of  the  growth  is  essentially  that  of 
microspira  comma,  though  at  times  it  is  a  little  more 
rapid  in  its  progress. 

GROWTH  ON  AGAR-AGAR. — On  meat-infusion  agar- 
agar,  neutral  or  slightly  alkaline  to  phenolphthalein, 
growth  is  very  rapid  at  the  body  temperature.  The 
general  character  of  the  growth  corresponds  to  that  of 
microspira  comma. 

BLOOD  SERUM. — The  growth  on  blood  serum,  after 
twenty-four  hours  at  body  temperature  appears  as  a  line 
of  depression,  which  increases  as  a  track  of  liquefaction, 
and  later  results  in  the  more  or  less  complete  liquefac- 
tion of  the  medium. 

BOUILLON. — Bouillon  becomes  uniformly  clouded  in 


508  BACTERIOLOGY. 

twenty-four  hours  at  the  body  temperature.  Its  reac- 
tion becomes  more  alkaline  as  growth  progresses.  A 
pellicle,  at  first  delicate,  later  denser  always  character- 
izes the  growth  in  this  medium. 

POTATO. — Usually  no  visible  growth. 

LITMUS  MILK. — In  fresh  litmus  milk  a  slight  degree 
of  acidity  is  noticed  after  twenty-four  hours  at  body 
temperature.  After  forty-eight  hours  this  acidity  is 
slightly  greater,  and  at  times  the  milk  shows  evidences 
of  coagulation,  though  not  always. 

BIOCHEMIC  CHARACTERS. — Microspira  Schuylkilli- 
ensis  is  an  aerobic,  facultative  organism.  In  fluid  media 
under  an  atmosphere  of  carbon  dioxide  in  sealed  tubes 
no  growth  is  observed. 

The  organism  grows  most  luxuriantly  at  about  37.5° 
C.  Growth  is  hardly  perceptible  at  10°  C.  It  is 
destroyed  by  an  exposure  of  five  minutes  to  50°  C. 

None  of  the  carbohydrates  are  broken  up  with  the 
liberation  of  gas. 

It  produces  indol  and  at  the  same  time  reduces 
nitrates  to  nitrites. 

PATHOGENESIS. — The  pathogenic  properties  of  this 
organism  are  best  seen  in  guinea-pigs  and  pigeons,  both 
of  which  are  uniformly  susceptible.  Rabbits  and 
chickens  resist  relatively  large  doses.  Mice  are  infected 
with  small  doses  injected  subcutaneously. 

The  most  characteristic  lesions  follow  the  injection  of 
cultures  into  the  pectoral  muscles  of  pigeons.  At  death 
the  inoculated  muscle  is  swollen,  necrotic,  and  the  over- 
lying tissues  are  redematous.  The  bacteria  are  found  in 
large  numbers  in  the  vicinity  of  the  seat  of  the  inocu- 
lation, and  in  relatively  small  numbers  in  the  blood  and 
internal  organs. 


MICROSPIRA   SCH UYLKILLIENSIS. 


509 


NOTE. — Since  the  epidemic  of  cholera  in  Hamburg 
quite  a  number  of  curved  or  spiral  organisms,  somewhat 
like  microspira  comma,  have  been  discovered.  For  the 
descriptions  of  these  the  reader  is  referred  to  current 
bacteriological  literature. 


CHAPTER    XXIII. 

Study  of  bacterium  anthracis,  and  of  the  effects  produced  its  by  inocula- 
tion into  animals — Peculiarities  of  the  organism  under  varying 
conditions  of  surroundings — Anthrax  vaccines — Anthrax  immune 
serum. 

THE  discovery  that  the  blood  of  animals  suffering 
from  splenic  fever,  or  anthrax,  always  contained  minute 
rod-shaped  bodies  (Pollender,  1855;  Davaine,  1863),  led 
to  a  group  of  investigations  that  have  not  only  fully  famil- 
iarized us  with  the  nature  of  this  malady  in  particular, 
but  have  perhaps  contributed  more  incidentally  to  our 
knowledge  of  bacteriology  in  general  than  studies  upon 
any  other  single  infective  process  or  its  causative  agent. 

The  direct  outcome  of  these  investigations  is  that  a 
rod-shaped  micro-organism,  now  known  as  bacterium  an- 
thracis, is  always  present  in  the  blood  of  animals  suffer- 
ing from  this  disease  ;  that  this  organism  can  be  obtained 
from  the  tissues  of  these  animals  in  pure  cultures ;  and 
that  these  artificial  cultures  of  bacterium  anthracis  when 
introduced  into  the  bodies  of  susceptible  animals  can 
again  produce  a  condition  identical  with  that  found  in 
the  animal  from  which  they  were  obtained.  The  dis- 
ease is  a  true  septicaemia,  and  after  death  the  capil- 
laries throughout  the  body  are  always  found  to  contain 
the  typical  rod-shaped  organism  in  larger  or  smaller 
numbers. 

This  organism,  when  isolated  in  pure  culture,  is  a 
bacterium  which  varies  considerably  in  length,  ranging 
from  short  rods,  2  to  3  //  in  length,  to  longer  threads, 
20  to  25  p.  in  length.  In  breadth  it  is  from  1  to 

510 


BACTERIUM  ANTHRAC1S.  511 

1.25  p.     Frequently  very  long   threads,  made    up   of 
several  rods  joined  end  to  end,  are  seen. 

When  obtained  directly  from  the  body  of  an  animal 
it  is  usually  in  the  form  of  short  rods  square  at  the  ends. 
If  highly  magnified,  the  ends  are  seen  to  be  a  trifle 
thicker  than  the  body  of  the  cell  and  somewhat  indented 
or  concave,  peculiarities  that  help  to  distinguish  it  from 
certain  other  organisms  that  are  somewhat  like  it  mor- 
phologically. (See  Fig.  81.) 

FIG.  81. 


Bacterium  anthracis,  highly  magnified  to  show  swellings  and  concavities  at 
extremities  of  the  single  cells. 

When  cultivated  artificially  at  the  temperature  of  the 
dy  the  bacterium  of  anthrax  presents  a  series  of  very 
interesting  stages. 

The  short  rods  develop  into  long  threads,  which 
my  be  seen  twisted  or  plaited  together  like  ropes, 
ich  thread  being  marked  by  the  points  of  juncture 
)f  the  short  rods  composing  it.  (Fig.  82,  a  and  b.) 
In  this  condition  it  remains  until  alterations  in  its  sur- 
roundings, the  most  conspicuous  being  diminution  of  its 
nutritive  supply,  favor  the  production  of  spores.  When 
this  stage  begins  changes  in  the  protoplasm  of  the 
bacteria  may  be  noticed  ;  they  become  marked  by  irregu- 
lar granular  bodies,  which  eventually  coalesce  into 
glistening  oval  spores,  one  of  which  lies  in  nearly  every 
segment  of  the  long  thread,  and  gives  to  the  thread  the 


512  BACTERIOLOGY. 

appearance  of  a  string  of  glistening  beads.  (Fig.  83.) 
In  this  stage  they  remain  but  a  short  time.  The  chains 
of  spores,  which  are  held  together  by  the  remains  of 

FIG.  82. 


Bacterium  anthraci*.    Plaited  and  twisted  threads  seen  in  fresh  growing 
cultures.    X  about  400  diameters. 

the  cells  in  which  they  formed,  become  broken  up,  and 
eventually  nothing  but  free  oval  spores,  and  here  and 
there  the  remains  of  mature  bacilli  which  have  under- 
gone degenerative  changes,  can  be  found.  In  this  con- 
dition the  spores,  capable  of  resisting  deleterious  influ- 

FlG.  83. 


Threads  of  bacterium  anthracis  containing  spores.    X  about  1200  diameters. 

1 

ences,  remain  and,  unless  their  surroundings  are  altered, 
continue  in  this  living,  though  inactive,  condition  for 
a  very  long  time.  If  again  placed  under  favorable 
conditions,  each  spore  will  germinate  into  a  mature  cell, 


BACTERIUM  AN  THE  A  CIS. 


513 


and  the  same  series  of  changes  will  be  repeated  until 
the  surroundings  become  again  gradually  unfavorable 
to  development,  when  spore-formation  again  takes  place. 
Spore-formation  occurs  only  at  temperatures  ranging 
from  18°  to  43°  C.,  37.5°  C.  being  the  optimum. 
Under  12°  C.  they  are  not  formed.  This  organism 
does  not  form  spores  in  the  tissues  of  the  living 
animal,  its  usual  condition  at  this  time  being  that  of 
short  rods ;  occasionally,  however,  somewhat  longer 
forms  may  be  seen. 

The  bacterium  of  anthrax  is  not  motile. 

GROWTH  ON  AGAR-AGAR. — Colonies  of  this  organ- 
ism, as  seen  upon  agar-agar,  present  a  very  typical 
appearance,  from  which  they  have  been  likened  unto 
the  head  of  Medusa.  From  a  central  point  which  is 

FIG.  84. 


•    Colony  of  bacterium  anthracis  on  agar-agar. 

more  or  less  dense,  consisting  of  a  felt-like  mass  of 
long  threads  irregularly  matted  together,  the  growth 
continues  outward  upon  the  surface  of  the  agar-agar. 
(Fig.  84.)  It  is  made  up  of  wavy  bundles  in  which 
the  threads  are  seen  to  lie  parallel  or  are  twisted  in 
strands  like  those  of  a  rope  ;  sometimes  they  have  a 
plaited  arrangement.  (See  Fig.  82.)  These  bundles 
twist  and  cross  in  all  directions,  and  eventually  dis- 

33 


514  BACTERIOLOGY. 

appear  at  the  periphery  of  the  colony.  At  the  ex- 
treme periphery  of  the  colonies  it  is  sometimes  possible 
to  trace  single  bundles  of  these  threads  for  long  dis- 
tances across  the  surface  of  the  agar-agar.  The  colony 
itself  is  not  circumscribed  in  appearance,  but  is  more 
or  less  irregularly  fringed  or  ragged,  or  scalloped.  To 
the  naked  eye  they  look  very  much  like  minute  pellicles 
of  raw  cotton  that  have  been  pressed  into  the  surface 
of  the  agar-agar. 

As  the  colonies  continue  to  grow  they  become  more 
and  more  dense  and  opaque,  and  granular  and  rough  on 
the  surface.  When  touched  with  a  sterilized  needle 
one  experiences  a  sensation  that  suggests  somewhat 
their  matted  structure.  They  are  never  moist  or 
creamy.  The  bit  that  is  taken  up  with  the  needle  is 
always  more  or  less  ragged. 

GELATIN. — The  colonies  on  gelatin  at  the  earliest 
stages  also  present  the  same  wavy  appearance ;  but  tins 
characteristic  soon  becomes  in  part  destroyed  by  the 
liquefaction  of  the  gelatin  which  is  produced  by  the 
growing  organisms.  This  allows  them  to  sink  to  the 
bottom  of  the  fluid,  where  they  lie  as  an  irregular  mass. 
Through  the  fluid  portion  of  the  gelatin  may  be  seen 
small  clumps  of  growing  bacteria,  which  look  very  much 
like  bits  of  cotton- wool. 

BOUILLON. — In  bouillon  the  growth  is  characterized 
by  the  formation  of  flaky  masses,  which  also  have  very 
much  the  appearance  of  bits  of  raw  cotton.  Micro- 
scopic examination  of  one  of*  these  flakes  reveals  the 
twisted  and  plaited  arrangement  of  the  long  threads, 

POTATO. — On  this  medium  it  develops  rapidly  as  a 
dull,  dry,  granular,  whitish  mass,  which  is  more  or  less 
limited  to  the  point  of  inoculation.  On  potato,  at  the 


BACTERIUM  ANTHRACIS.  515 

temperature  of  the  incubator,  spore-formation  may  be 
easily  observed. 

STAB-  AND  SLANT-CULTURES. — Stab-  and  slant-cult- 
ures on  agar-agar  present  in  general  the  appearances 
given  for  the  colonies,  except  that  the  growth  is  much 
more  extensive.  The  growth  is  always  more  pro- 
nounced on  the  surface  than  down  the  track  of  the 
needle. 

On  gelatin  it  causes  liquefaction,  which  begins  on  the 
surface  at  the  point  inoculated  and  spreads  outward  and 
downward. 

It  grows  best  with  access  to  oxygen,  and  very  poorly 
when  the  supply  of  that  gas  is  interfered  with. 

Under  favorable  conditions  of  aeration,  nutrition,  and 
temperature  its  growth  is  rapid. 

Under  12°  C.  and  above  45°  C.  no  growth  occurs. 
Its  optimum  temperature  is  that  of  the  human  body, 
viz.,  37°-38°  C. 

The  spores  of  bacterium  anthracis  are  very  resistant  to 
heat,  though  the  degree  of  resistance  varies  with  spores 
of  different  origin.  Von  Esmarch  found  that  anthrax 
spores  from  some  sources  were  readily  killed  by  an  ex- 
posure of  one  minute  to  the  temperature  of  steam, 
whereas  spores  from  other  sources  resisted  this  temper- 
ature longer,  in  some  cases  as  long  as  twelve  minutes. 

STAINING. — Anthrax  bacteria  stain  readily  with  the 
ordinary  aniline  dyes.  In  tissues  their  presence  may 
also  be  demonstrated  by  the  ordinary  aniline  stain- 
ing-fluids  or  by  Gram's  method.  They  may  also  be 
stained  in  tissues  with  a  strong  watery  solution  of 
dahlia,  after  which  the  sections  are  decolorized  in  2  per 
cent,  sodium  carbonate  solution,  washed  in  water,  dehy- 
drated in  alcohol,  cleared  in  xylol,  and  mounted  in  bal- 


516  BACTERIOLOGY. 

sam.  This  leaves  the  bacilli  stained,  while  the  tissues 
containing  them  are  decolorized  ;  or  the  latter  may  be 
stained  a  contrast-color — with  eosin,  for  example — after 
dehydration  in  alcohol  and  before  clearing  in  xylol.  In 
this  case  they  must  be  washed  again  in  alcohol  before 
using  the  xylol.  In  a  preparation  treated  in  this  way 
the  rod-shaped  organisms  are  of  a  purple  color,  and  will 
be  seen  in  the  capillaries  of  the  tissues,  while  the  tissues 
themselves  are  of  a  pale  rose  color. 

INOCULATION  INTO  ANIMALS. — Introduce  into  the 
subcutaneous  tissues  of  the  abdominal  wall  of  a  guinea- 
pig  or  rabbit  a  portion  of  a  pure  culture  of  bacterium 
anthracis.  The  animal  usually  succumbs  in  from  thirty- 
six  to  forty-eight  hours.  Little  or  no  reaction  at  the 
immediate  point  of  inoculation  will  be  noticed ;  but 
beyond  this,  extending  for  a  long  distance  over  the 
abdomen  and  thorax,  the  tissues  will  be  markedly 
redematous.  Here  and  there,  scattered  through  this 
oedematous  tissue,  small  ecchymoses  will  be  seen.  The 
underlying  muscles  are  pale  in  color.  Inspection  of  the 
internal  viscera  reveals  no  very  marked  macroscopic 
changes  except  in  the  spleen.  This  is  enlarged,  dark 
in  color,  and  soft.  The  liver  may  present  the  appear- 
ance of  cloudy  swelling ;  the  lungs  may  be  red  or  pale 
red  in  color ;  the  heart  is  usually  filled  with  blood.  No 
other  changes  can  be  seen  by  the  naked  eye. 

Prepare  cover-slip  preparations  from  the  blood  and 
other  viscera.  They  will  all  be  found  to  contain  short 
rods  in  large  numbers.  Nowhere  can  spore-formation 
be  detected.  Upon  microscopic  examination  of  sec-  * 
tions  of  the  organs  which  have  been  hardened  in 
alcohol  the  capillaries  are  seen  to  be  filled  with  -the 
bacteria ;  in  some  places  closely  packed  in  large  num- 


BACTERIUM  ANTHRACIS.  517 

bers,  at  other  points  fewer  in  number.  Usually 
they  are  present  in  largest  numbers  in  those  tis- 
sues having  the  greatest  capillary  distribution  and  at 
those  points  at  which  the  circulation  is  slowest.  They 
are  uniformly  distributed  through  the  spleen.  The 
glomeruli  of  the  kidneys  and  the  capillaries  of  the 


\ 


FIG.  85. 

\  V; 

v  , 

V  ^ 

*>\\ 

v\ 

w         V 

^\                        Vv        \ 

^     *l 

V               x 

\ 

v    N 

V 

\  Sto 
v\  y  VN 

\ 

Bacterium  antkracis  in  liver  of  mouse.    X  about  450  diameters.    Bacteria 
stained  by  Gram's  method  ;  tissue  stained  with  Bismarck-brown. 

lungs  are  frequently  packed  with  them.  The  capillaries 
of  the  liver  contain  them  in  large  numbers.  (Fig.  85.) 
Hemorrhages,  probably  due  to  rupture  of  capillaries 
by  the  mechanical  pressure  of  the  bacteria  which  are 
developing  within  them,  not  uncommonly  occur. 
When  these  occur  in  the  mucous  membranes  of  the 
alimentary  tract  the  blood  may  escape  through  the 
mouth  or  anus ;  when  in  the  kidneys,  through  the  uri- 
niferous  tubules. 

Cultures  from  the  different  organs  or  from  the  oederna- 
tous  fluid  about  the  point  of  inoculation  result  in  growth 
of  bacterium  anthracis. 

The  amphibia,  dogs,  and  the  majority  of  birds  are 
not  susceptible  to  this  disease.  Rats  are  difficult  to 


MM  /.'  H'TKItHH.iHJ  I 

illlri'l,  KllUlilw,  ^IlilK'll  piy,H,  Nvllilr  lilier,  e  I'M  V  llUllHC 
Illicc,  HlMM'pi  Mini  rnlllc  ii-  HIIHCt'pl  ihlc,  Infer!  ion  HIM.)' 
•••  •  HI  either  llinni(.'li  Mi.  ciiviiln!  imi,  ilu-.u  I.  il,  uir 

pllltnilJM'M,    I  lll'llllj'll     |||<>     ||  I  imt'll  1.111')'    ll'IM'l,   Ml',    MM     N\e     llMAe 

jilHt  Neen,  lliroii^li  (lie  HIlbeillHIieoiiH  liMHiii'H. 


I'lHH  i;<  Ti\  i;    INumi.A'l  ION, 

Tin1    iiinf.l    ii(il<'\\orl  liv   ii|>|>liriil  inn    nC  ni'l  ili(Miill\    prr 
I  ii  I  I'ci  I    living    Nin-i'iiM'M    i"    ili<      prnliM'limi    --I     iiniiniilh 
M^MIIIH!    in  Ircl  inn   JM    HIMMI    in    omiiirrlion    \\illi    nnllirnx 
in   H|I»M'|»  Mini   in   liovincH, 

'•      M    Niirit'lv    "i    promuhircH    (In*    virulent    MiillmiN 

lllli'lcrilllll   IllMV    !M<  ill   purl   nr   Inlnllv    I'M|»|HM|  nf   itn   (Mlllin 

H'lMiii'  |im|M<rtifH,      1  1    IM  llimii^li   tli<<  vn-v  mild  miiih 
Inlinniil  (liMlnt'liMiKM*  INIIIMIM!    in  uniinalM  VIH-.  m.ii.  .1  wild 
HiK'li  Wt»llK»Mii((l  iMillnroM  lluil  |»ri»l(M'l  ion  in  ol'lcn  MlVurdrd 
M^M)IIH(  llio  Ht»v«M'«»r,  htMjiKMillv  llii.il    lunn  «»!'  (lie   mi-. 
linn, 

Without  reviewing  Ihe  vnriniiM  inelltodH  Mini  luive 
|MM«II  cinploviMl  I'nr  iiMrniin!  iti^  (he  \iriilenee  oC  (Inn 
orgnnlHiii  In  M  dogitm  HiiiluMe  lor  prnleeliv(«  YIKMMIIM 
(inn,  il  will  miillloo  In  HMV  Mini  I  lie  nmsl  HMlinliu'lorv 
h'HiilU  IIMVP  IUMMI  olilniiiiMl  l»y  Inn^1  rnnliniMMl  riillivn 
(inn  (Icn  In  thirty  dnyn)  Ml  n  leinpenHnre  nf  i'rnni  '12° 
to  l.'t"  (\  In  lliin  phMM'dnro  lite  npore  free,  virnlont 
/»iM'/iT/»iw  ifjiMnf.'/N,  ohlMined  diiveilN  In  mi  llir  hlnnd  nf 
n  itM'eiilly  dond  Miiiiunl,  in  hroii^ht  al  once  into  sterile 
nnlrient  bouillon  in  uhont  twenty  leMMnhes,  wliieh  ure 
immedlMlely  plneed  in  nn  inenhntor  that  in  enrelnlls 
it^uliHiMl  to  innintnin  M  lempenitnre  of  l^,r>°  (  '.  There 
1  1.  -i,  i,  i  not,  ho  a  IliuMiiMlion  of  over  ().  l(>  (\ 

Al^er  nhont  M  \veeK  M  tnhe  in  r<(ino\'ed  from  I  he  inon- 
hntor  on  oiioli  nnoot^Hivo  du    niul  its  virnhMioo  levied  at 


.....  •    on   imnmH,     'Tin    .I."!..    ,.t    ;IM.  minimi!   <>s|>.  M 

,  n,,.ii.\  the  ovilturti  grown  undo  i  HI.  .  ,  n, 


IH  (III.  i  mm,.  |  |»y  ll'MtH  ll|inil  I'llllhilH,  ^llilll«l|  |li^H,  Mild 
.....  10,  Till'  Ill'Ml  •  nil.  in  i.  m.-\  -  .1  111.  IN  ,,|  m  i\  Mill 

Kill    ruUiilM,    lln'    inoHt    n'MiMlmii    «|    il,,     M,,.. 

IIHnI   Inr  lln<  Irnl,  \\lul.'  il    \\ill   .•nlmnlx    I  .11   tin- 

l-i       m.  I  mi.,       id.  i    m.  -Mi,  i  i\\,,  ..i  i  Invr  tin  VH  rnhlillH 

\\ill    il"    Inn^'fl1    Hllrrilinl.   In     I  .....  ill  ill.  .11    will,    |||,      ,  ullui. 

I  i    I      i-   ui"\  '  'I     I  .....  i    II"       in-   nl-  il.'l  .    \\  Illlr     ll.>     -  li  mm.  id.  •  n 

\\ill  KM  vH  h<»  noticed  in  iln  piHlio^'oncHiH  Inr  llm  ml.,i 
l\\«>  MJMMMCH.  Aflrl'  ft  MI  r  In  HOVCIl  <|IIVH  innro  ii(Millm< 
HUN  I"  -I  ......  nl.  -M.l  lli.il  Killti  niily  mi.  .-.  Mi.-  ^illllOH- 

pi^'H    <'H('ll|>iii.-    .     \\lnl.      iilhmil.lv.il     III.      .    •  |M  i  .......  I     I.. 

.  MM!  iiiucil,  ||     ili-i.rrr    nl'   lllli'lllllll  Kill     Illliy    I"      i.    I'll-.  I    111 

\\llhlllll.        .iM'llllinlll      llMH    IM'I       .        .Mill.       |)(IW(M       "I 

II  Illnll    r,    Ilinllg'll    il    hllll    ivlnill'.     ||M    villllllv  I  II  V»'Hl  1 
li.'M     nl       llir.r      II  I  I  I'll  I  III  I  i<>||H     nllOWK     ill'   in      <<>      |  niMHl'HH      II  1  1 
ill''     .'lllini.'lc|'ih|i('h    (>!'    .  ill.  .  LI.  .  I     ml  lii   i  ,    IM.I.MI,     il,,   • 
"i..\\        I..\\!N     mill     |rt«M     vipnrmiiily     \\li-n     I  I'llllMpllllil'  •  I. 
I  IK   \     *  l<  i     i  M  1  1      l'»  in     MjHin-M    \\  I  .....       |...    .  .1    In    ||    I  ii}>  1  1    I.  m 

jxiilni,  in.  I     i  .......  KIOplOAlly     lli«-N      |»l.     .nl      -   \  I'l.'IKM'H 

nl'     ilrm-||rnill..n  \\    In  n     1  1  .  1  I  ...  I  .....  I      I  .......  I.     lln          I    m 

I'    iiiiiniiilt'.    Iliry    dif  .  i  .....  il.      I.  nl    ;  hj'lills     I»MI»M<|    Mi. 
nilc    nl     n  ......  liilimi,    iin.  I    <!  ......  I.     i        i    inli',    riniMi     ili< 

^'I'lM-nil     !.r|illi-|i-IIIIH     Illlll      iMTlll-H     III     HIIHri'jilil-l  .....  llllllH 

HI.  i  n  ......  1.  1  1  1..  1  1  \\  iiK  i  M.I  m  1  1  .  n  1  1  n  i  .    ..i  iiiin  ofgnnliRii 

III  III.  |.i  i.  I  i.  .1  •  ni|i|..\  m.  nl  ..I  llirHO  ||,M,I'IIIIM  I  '  'I  '  nil 
IMVH  Inr  |>rn|rr|ivi'  |>lir|inH('H  I  \\  "  ..............  |'l"  ••' 

I  I,.    ,     u.  ,,      .1,    |gn  .i,  -I    I.       I1  .  i.  n,     MM    "  llrnl."     ",.l 
.....  I         .......          I  h.       i.i'Hl."  in  Hi'   --M-    ill  ii  I  .11-  -I 

"III  I  I  ...........      I  II,         |,l  <     llll.MI    I)    N       I.        I  \\    Illh        III. 

nil.  I    "    I         II,  il     vv  1,1.    I,      I    ,11,  ,1      l..,||,      ,  .........  I      •>  .......     jliu'H, 

I.  ill     I.  ill.  .1     In      1,11      ||,,        i    iM.il  \\   lim     liii  .....  mm  ,1    , 


520  BACTERIOLOGY. 

such  as  sheep  or  cattle,  are  to  be  protected  by  vaccina- 
tion with  these  vaccines,  a  subcutaneous  inoculation  of 
about  0.3  c.c.  of  the  first  vaccine  is  usually  given. 
This  should  be  practically  without  noticeable  effect, 
causing  neither  rise  of  body-temperature  nor  other 
constitutional  or  local  symptoms.  After  a  period  of 
about  two  weeks  the  second  vaccine  is  injected  in  the 
same  way  ;  this  may  or  may  not  cause  disturbance. 
In  the  event  of  its  doing  so  the  symptoms  are  rarely 
alarming,  and,  if  the  vaccines  have  been  properly  pre- 
pared and  tested  before  use,  they  disappear  within  a 
short  time  after  the  injection. 

In  the  large  majority  of  cases  sheep,  bovines,  horses, 
and  mules  may  be  safely  protected  against  anthrax  by 
the  careful  practice  of  this  method. 

Sobernheim l  found  that  it  was  possible  to  bring  about 
a  high  degree  of  immunity  against  bacterium  anthracis 
by  means  of  the  vaccines  1  and  2  of  Pasteur,  with  sub- 
sequent inoculations  of  virulent  organisms.  He  employed 
the  serum  of  animals  thus  immunized  in  the  treatment  of 
sheep  that  had  been  injected  with  highly  virulent  anthrax 
bacteria.  Five  sheep  were  treated  in  this  way,  and  all 
of  them  recovered  with  only  slight  rise  in  temperature  and 
more  or  less  marked  infiltration  at  the  point  of  injection, 
while  control  animals  died  very  promptly. 

Sobernheim 2  reports  an  improvement  on  the  method 
of  protective  inoculation  against  anthrax  in  which  he 
uses  a  combination  of  anthrax  vaccines  and  immune 
serum,  in  which  the  results  are  far  more  satisfactory 
than  with  the  anthrax  vaccines  alone.  He  states  that 
this  new  method  has  the  following  advantages  over  the 

1  Sobernheim  :  Berliner  klin.  Wochenschr.,  1897. 

2  Ibid.,  1902,  p.  516. 


BACTERIUM  ANTHRACIS.  521 

Pasteur  method  :  (1)  That  the  immunization  can  be  car- 
ried out  without  losing  any  of  the  animals ;  (2)  that  it 
can  be  completed  in  one  day  ;  (3)  that  stronger  and  more 
active  cultures  can  be  employed  and  therefore  a  more 
durable  immunity  obtained;  and  (4)  that  the  serum  alone 
can  be  employed  as  a  curative  agent. 

Schlemmer  employed  the  Sobernheim  method  on  39 
oxen  in  which  the  results  were  not  uniformly  satis- 
factory, as  one  of  the  animals  became  ill  and  most  of 
them  showed  febrile  reactions.  Schlemmer  attributes 
the  unfavorable  results  to  the  small  dose  of  immune 
serum  employed,  namely,  10  cubic  centimetres,  and 
believes  that  for  large  animals  larger  doses  of  the 
immune  serum  should  be  used. 

EXPERIMENTS., 

Prepare  three  cultures  of  bacterium  anthracis — one 
upon  gelatin,  one  upon  agar-agar,  and  one  upon  potato. 
Allow  the  gelatin  culture  to  remain  at  the  ordinary 
temperature  of  the  room,  place  the  agar-agar  culture 
in  the  incubator,  and  the  potato  culture  at  a  temper- 
ature not  above  18°  to  20°  C.  Prepare  cover-slips 
from  each  from  day  to  day.  What  differences  are 
observed  ? 

Prepare  two  potato  cultures  of  bacterium  anthra- 
cis. Place  one  in  the  incubator  and  maintain  the 
other  at  a  temperature  of  from  18°  to  20°  C.  Ex- 
amine them  each  day.  Do  they  develop  in  the  same 
way  ? 

From  a  fresh  culture  of  bacterium  anthracis,  in  which 
spore-formation  is  not  yet  begun  (which  is  the  -surest 


522  &ACTERIOLOG  Y. 

source  from  which  to  obtain  non-xpore-bearing  anthrax 
bacteria),  prepare  a  hanging-drop  preparation  ;  also  a 
cover-slip  preparation  in  the  usual  way  and  stain  it 
with  a  strong  gentian-violet  solution ;  and  another 
cover-slip  preparation  which  is  to  be  drawn  through 
a  flame  twelve  to  fifteen  times,  stained  with  aniline 
gentian-violet,  washed  in  iodine  solution  and  then 
in  water.  Examine  these  microscopically.  Do  they 
all  present  the  same  appearance?  To  what  are  the 
differences  due? 

Do  the  anthrax  threads,  as  seen  in  a  fresh,  growing, 
hanging  drop,  present  the  same  morphological  appear- 
ance as  when  dried  and  stained  upon  a  cover-slip? 
How  do  they  differ? 

Liquefy  a  tube  of  agar-agar,  and  when  it  is  at  the 
temperature  of  40°  to  43°  C.  add  a  very  minute  quan- 
tity of  an  anthrax  culture  which  is  far  advanced  in  the 
spore-stage.  Mix  it  thoroughly  with  the  liquid  agar- 
agar  and  from  this  prepare  several  hanging  drops  under 
strict  antiseptic  precautions,  using  the  fluid  agar-agar 
for  the  drops  instead  of  bouillon  or  salt-solution.  Select 
from  among  these  preparations  that  one  in  which  the 
smallest  number  of  spores  are  present.  Under  the 
microscope  observe  the  development  of  a  spore  into  a 
mature  cell.  Describe  carefully  the  developmental 
stages. 

Prepare  a  1  : 1000  solution  of  carbolic  acid  in  bouil- 
lon. Inoculate  this  with  virulent  anthrax  spores.  If 
no  development  occurs  after  two  or  three  days  at  the 
temperature  of  the  thermostat,  prepare  a  solution  of 
1 : 1200,  and  continue  until  the  point  is  reached  at 


BACTERIUM  ANTI&ACLS.  523 

which  the  amount  of  carbolic  acid  present  just  permits 
of  the  development  of  the  spores.  When  the  proper 
dilution  is  reached  prepare  a  dozen  of  such  tubes  and 
inoculate  one  of  them  with  virulent  anthrax  spores. 
As  soon  as  development  is  well  advanced  transfer  a 
loopful  from  this  tube  into  a  second  of  the  carbolic  acid 
tubes ;  when  this  has  developed,  then  from  this  into  a 
third,  etc.  After  five  or  six  generations  have  been 
treated  in  this  way  study  the  spore-production  of  the 
organisms  in  that  tube.  If  it  is  normal,  continue  to 
inoculate  from  one  carbolic  acid  tube  to  another,  and 
see  if  it  is  possible  by  this  means  to  influence  in  any 
way  the  production  of  spores  by  the  organism  with 
which  you  are  working.  What  is  the  effect,  if  any? 

Prepare  two  bouillon  cultures,  each  from  one  drop  of 
blood  of  an  animal  dead  of  anthrax.  (Why  from  the 
blood  of  an  animal  and  not  from  a  culture  ?)  Allow  one 
of  them  to  grow  for  from  fourteen  to  eighteen  hours  in 
the  incubator ;  allow  the  other  to  grow  at  the  same  tem- 
perature for  three  or  four  days.  Remove  the  first  tube 
after  the  time  mentioned  and  subject  it  to  a  temperature 
of  80°  C.  for  thirty  minutes.  At  the  end  of  this  time 
prepare  four  plates  from  it.  Make  each  plate  with  one 
drop  from  the  heated  bouillon  culture.  At  the  end  of 
three  or  four  days  treat  the  second  tube  in  identically 
the  same  way.  How  do  the  number  of  colonies  which 
develop  from  the  two  cultures  compare  ?  Was  there 
any  difference  in  the  time  required  for  their  develop- 
ment on  the  plates? 

From  a  potato  culture  of  bacterium  anthracis  which 
has  been  in  the  incubator  for  three  or  four  days  scrape 


524  BACTERIOLOGY. 

away  the  growth  and  carefully  break  it  up  in  10  c.c. 
of  sterilized  physiological  salt-solution.  The  more 
thoroughly  it  is  broken  up  the  more  accurate  will  be  the 
results  of  the  experiment.  Place  this  in  a  bath  of  boil- 
ing water,  and  at  the  end  of  one,  three,  five,  seven,  and 
ten  minutes  make  plates  upon  agar-agar  each  with  one 
loopful  of  the  contents  of  this  tube.  Are  the  results 
on  the  plates  alike  ? 

Determine  the  exact  time  necessary  to  sterilize  ob- 
jects, such  as  silk  or  cotton  threads,  on  which  anthrax 
spores  have  been  dried,  by  the  steam  method  and  by 
the  hot-air  method. 

Prepare  a  bouillon  culture  from  the  blood  of  an  ani- 
mal just  dead  of  anthrax.  After  this  has  been  in  the 
incubator  for  from  three  to  four  hours  subject  it  to  a 
temperature  of  55°  C.  for  ten  minutes.  At  the  end 
of  this  time  make  plates  from  it  and  also  inoculate  a 
rabbit  subcutaneously  with  it.  What  are  the  results? 
Are  the  colonies  on  the  plates  in  every  way  charac- 
teristic ? 

Inoculate  six  Erlenmeyer  flasks  of  sterile  bouillon, 
each  containing  about  35  c.c.  of  the  medium,  from 
either  the  blood  of  an  animal  just  dead  of  anthrax  or 
from  a  fresh  virulent  culture  in  which  no  spores  are 
formed. 

Place  these  flasks  in  the  incubator  at  a  temperature 
of  42.5°  C.  At  the  end  of  five,  ten,  fifteen,  twenty, 
twenty-five,  or  more  days  remove  a  flask.  Label 
each  flask  as  it  is  taken  from  the  incubator  with  the 
exact  number  of  days  that  it  has  been  at  the  tempera- 


BACTERIUM  ANTHRACIS.  525 

ture  of  42.5°  C.  Study  each  flask  carefully,  both  in 
its  culture-peculiarities  and  in  its  pathogenic  properties 
when  employed  on  animals. 

Are  these  cultures  identical  in  all  respects  with  those 
that  have  been  kept  at  37°  C.? 

If  they  differ,  in  what  respect  is  the  difference  most 
conspicuous  ? 

Should  any  of  the  animals  survive  the  inoculations 
made  from  the  different  cultures  in  the  foregoing  ex- 
periment, note  carefully  which  one  it  is,  and  after  ten 
to  twelve  days  repeat  the  inoculation,  using  the  same 
culture;  if  it  again  survives,  inoculate  it  with  the  cult- 
ure preceding  the  one  just  used  in  the  order  of  removal 
from  the  incubator ;  if  it  still  survives,  inoculate  it  with 
virulent  anthrax.  What  is  the  result?  How  is  the 
result  to  be  explained  ?  Do  the  cultures  which  were 
made  from  these  flasks  at  the  time  of  their  removal 
from  the  incubator  act  in  the  same  way  toward  animals 
as  the  organisms  growing  in  the  flasks  ?  Is  the  action 
of  each  of  these  cultures  the  same  for  mice,  guinea-pigs, 
and  rabbits? 

Prepare  a  2  per  cent,  solution  of  sulphuric  acid  in 
distilled  water;  suspend  in  this  a  number  of  anthrax 
spores ;  at  the  end  of  three,  six,  and  nine  days  at  35°  C. 
inoculate  both  a  guinea-pig  and  a  rabbit.  Prepare 
cultures  from  this  suspension  on  the  third,  sixth,  and 
ninth  days ;  when  the  cultures  have  developed  inoculate 
a  rabbit  and  a  guinea-pig  from  the  culture  made  on  the 
ninth  day.  Should  the  animals  survive,  inoculate  them 
again  after  three  or  four  days  with  a  culture  made  on 
the  sixth  day.  Do  the  results  appear  in  any  way 
peculiar  ? 


526  BA  CTERIOL  0  G  Y. 


\  IMMUNE  SERUM.  —  Sanfelice1  experimented 
with  the  serum  of  dogs  that  had  been  immunized  from 
anthrax  bacteria.  This  serum  possessed  immunizing 
and  curative  properties,  as  shown  by  experiments  upon 
animals.  He  had  an  opportunity  of  trying  the  serum, 
with  favorable  results,  upon  a  man  who  had  contracted 
anthrax.  The  total  amount  of  serum  employed  was  56 
cubic  centimetres.  There  was  no  reaction  at  the  point 
of  injection  of  the  serum.  The  therapeutic  effect  of  the 
administration  of  serum  was  a  general  improvement  in 
the  symptoms,  marked  fall  of  the  temperature  on  the 
second,  and  complete  apyrexia  on  the  third,  day.  The 
effect  on  the  local  anthrax  lesion  manifested  itself  in 
reduction  and,  finally,  disappearance  of  the  oedema,  fol- 
lowed first  by  an  increased  swelling  of  the  glands,  which 
decreased  again  subsequently.  He  states  that  the  serum 
treatment  should  be  continued  not  only  till  the  temper- 
ature has  fallen  to  normal  and  a  diminution  of  the 
oadema  is  apparent,  but  also  until  there  is  marked  re- 
duction in  the  size  of  the  swollen  lymph-glands. 

Sclavo  2  immunized  a  number  of  animals,  principally 
sheep  and  goats,  with  the  two  vaccines  of  Pasteur,  fol- 
lowed by  repeated  injections  of  increasing  quantities  of 
virulent  cultures.  By  this  means  he  obtained  an  im- 
mune serum  wrhich  had  protective  as  well  as  curative 
properties  when  tested  upon  guinea-pigs  and  rabbits. 
He  found  that  this  serum  had  neither  bactericidal  nor 
antitoxic  properties. 

Cicognani3  employed  Sclavo'  s  immune  serum  on  12 
persons  suffering  from  various  grades  of  anthrax  infec- 

1  Sanfelice  :  Centralblatt  f.  Bacteriologie,  Originate,  1902,  Bd.  33. 

2  Sclavo:  Bulletin  de  1'Institut  Pasteur,  T.  I.,  1903,  p.  305. 

3  Cicognani:  Centralblatt  f.  Bacteriologie,  1902,  ref.  Bd.  31,  p.  725. 


ANTHRAX  IMMUNE  SERUM.  527 

tion,  some  of  the  cases  being  severe  infections  in  which 
the  prognosis  would  otherwise  have  been  very  unfavor- 
able. The  duration  of  the  disease  was  always  very 
much  shortened  and  all  recovered. 

Lazaretti 1  reports  23  cases  of  human  infection  with 
bacterium  anthracis  in  which  Sclavo's  immune  serum 
was  employed  with  recovery  in  each  case.  Another 
patient,  suffering  from  chronic  alcoholism  and  malaria, 
did  not  recover. 

1  Lazaretti :  Deutsche  Vierteljahrsschrift  f.  offeutliche  Gesuudheits- 
pflege,  1903,  Bd.  35,  Supplement,  p.  253. 


CHAPTER    XXIV. 

The  most  important  of  the  organisms  found  in  the  soil — The  nitrify- 
ing bacteria— The  bacillus  of  tetanus — The  bacillus  of  malignant 
oedema — The  bacillus  of  sj^mptomatic  anthrax — Bacterium  Welchii 
— Bacillus  sporogenes. 

THE    NITRIFYING    BACTERIA. 

BY  the  employment  of  bacteriological  methods  in 
the  study  of  the  soil  much  light  has  been  shed  upon 
the  cause  and  nature  of  the  interesting  and  momen- 
tous biological  phenomena  there  constantly  in  prog- 
ress. Of  these,  the  one  that  is  of  the  greatest  im- 
portance comprises  those  changes  that  accompany  the 
widespread  process  of  disintegration  and  decomposi- 
tion, to  which  reference  has  already  been  made.  (See 
Chapter  I.)  This  resolution  of  dead  complex  organic 
compounds  into  simpler  structures  that  are  assimilable 
as  food  by  growing  vegetation  is  dependent  upon 
the  activities  of  bacteria  located  in  the  superficial 
layers  of  the  ground.  It  is  not  a  simple  process, 
brought  about  by  a  single,  specific  species  of  bacteria, 
but  represents  a  series  of  metabolic  alterations,  each 
definite  step  of  which  is  most  probably  the  result  of 
the  activities  of  different  species  or  groups  of  species, 
acting  singly  or  together  (symbiotically).  Our  knowl- 
edge upon  the  subject  is  not  sufficient  to  permit  us  to 
follow  in  detail  the  manifold  alterations  undergone 
by  dead  organic  material  in  the  process  of  decomposi- 
tion that  results  in  its  conversion  into  inorganic  com-  " 
pounds,  with  the  formation  of  carbonic  acid,  ammonia, 
and  water  as  the  conspicuous  end-products.  It  suffices 

528 


THE  NITRIFYING  BACTERIA.  529 

to  say  that  wherever  dead  organic  matters  are  exposed 
to  the  action  of  the  great  group  of  saprophytic  bacteria, 
in  which  are  found  many  different  species,  the  altera- 
tions through  which  they  pass  are  ultimately  character- 
ized by  the  appearance  of  these  three  bodies.  When  the 
process  of  decomposition  occurs  in  the  soil,  however,  it 
does  not  cease  at  this  point,  but  we  find  still  further 
alterations  —  alterations  concerning  more  particularly 
the  ammonia.  This  change  in  ammonia  is  character- 
ized by  the  products  of  its  oxidation,  viz.,  by  the  for- 
mation of  nitrous  and  nitric  acids  and  their  salts ;  it  is 
not  a  result  of  the  direct  action  of  atmospheric  oxygen 
upon  the  ammonia,  but  occurs  through  the  instrumen- 
tality of  a  special  group  of  saprophytes  known  as  the 
nitrifying  organisms.  They  are  found  in  the  most  super- 
ficial layers  of  the  ground,  and  though  more  common 
in  some  places  than  in  others,  they  are,  nevertheless, 
present  over  the  entire  surface  of  the  earth.  The 
most  conspicuous  example  of  the  functional  activity  of 
this  specific  form  of  soil  organism  is  seen  in  the  im- 
mense saltpetre-beds  of  Chili  and  Peru,  where,  by  the 
activities  of  these  microscopic  plants,  nitrates  are  pro- 
duced from  the  ammonia  of  the  faecal  evacuations  of 
sea-fowl  in  such  enormous  quantities  as  to  form  the 
source  of  supply  of  this  article  for  the  commercial 
world.  A  more  familiar  example,  though  hardly  upon 
such  a  great  scale,  is  seen  in  the  decomposition  and 
subsequent  nitrification  of  the  organic  matters  of  sew- 
age and  other  impure  waters  in  the  process  of  puri- 
fication by  filtration  through  the  soil,  a  process  in  which 
it  is  possible  to  follow,  by  chemical  means,  the  organic 
matters  from  their  condition  as  such  through  their  con- 
spicuous modifications  to  their  ultimate  conversion  into 


530  HA  CTERIOL  OGY. 

ammonia,  nitrous  and  nitric  acids.  In  fact,  the  same 
breaking  down  and  building  up,  resulting  ultimately 
in  nitrification,  occurs  in  all  nitrogenous  matters  that 
are  deposited  upon  the  soil  and  allowed  to  decay.  It  is 
largely  through  this  means  that  growing  vegetation 
obtains  the  nitrogen  necessary  for  the  nutrition  of  its 
tissues,  and  when  viewed  from  this  standpoint  AVC  ap- 
preciate the  importance  of  this  process  to  all  life,  ani- 
mal as  well  as  vegetable,  upon  the  earth. 

These  very  important  and  interesting  nitrifying 
organisms,  of  which  there  appear  to  be  several,  have 
been  the  subject  of  much  study,  and  are  found  to 
possess  peculiarities  of  sufficient  interest  to  justify  a 
brief  description.  For  a  long  time  all  efforts  to  iso- 
late them  from  the  soils  in  which  they  were  believed 
to  be  present,  and  to  cultivate  them  by  the  processes 
commonly  employed  in  bacteriological  work,  resulted, 
in  failure ;  and  it  was  not  until  it  was  found  that 
the  ordinary  methods  of  bacteriological  research  were 
in  no  way  applicable  to  the  study  of  these  bacteria 
that  other,  and  ultimately  successful,  methods  were  de- 
vised. By  these  special  devices  nitrifying  bacteria, 
capable  of  oxidizing  ammonia  to  nitric  acid,  have  been 
isolated  and  cultivated,  and  the  more  important  of  their 
biological  peculiarities  recorded  by  Winogradsky  in 
Switzerland,  by  G.  C.  and  P.  F.  Frankland  in  Eng- 
land, and  by  Chester,  Jordan,  and  Richards  in  this 
country.  From  the  similarity  of  the  properties,  given 
by  these  several  observers,  of  the  nitrifying  organisms 
isolated  by  them,  it  seems  likely  that  they  have  all  been 
working  with  either  the  same  organism  or  very  closely 
allied  species. 

The  organism  generally  known  as  the  nitro-monas  of 


THE  NITRIFYING  BACTERIA.  531 

Winognidsky  is  a  short,  oval,,  and  frequently  almost 
spherical  cell.  It  divides  as  usual  for  bacteria,  but 
there  is  little  tendency  for  the  daughter-cells  to  adhere 
together  or  to  form  chains.  In  cultures  they  are  com- 
monly massed  together,  by  a  gelatinous  material,  in  the 
form  of  zoogloea.  They  do  not  form  spores,  and  are 
probably  not  motile,  though  Winogradsky  believes 
he  has  occasionally  detected  them  in  active  motion. 
As  has  been  stated,  they  do  not  grow  upon  ordinary 
nutrient  media,  and  cannot,  therefore,  be  isolated  by 
the  means  commonly  employed  to  separate  different 
species  of  bacteria.  The  most  astonishing  property  of 
this  organism  is  its  ability  to  grow  and  perform  its 
specific  fermentative  function  in  solutions  devoid  of 
organic  matter.  It  is  believed  to  be  able  to  obtain  its 
necessary  carbon  from  carbonic  acid.  For  its  isolation 
and  cultivation  Winogradsky  recommends  the  following 
solution  : 

Ammonium  sulphate 1  gramme. 

Potassium  phosphate 1 

Pure  water 1000  c.o. 

To  each  flask  containing  100  c.c.  of  this  fluid  is  added 
from  0.5  to  1  gramme  of  basic  magnesium  carbonate 
suspended  in  a  little  distilled  water  and  sterilized  by 
boiling.  One  of  the  flasks  is  then  to  be  inoculated  with 
a  minute  portion  of  the  soil  under  investigation,  and 
after  four  or  five  days  a  small  portion  is  to  be  with- 
drawn, by  means  of  a  capillary  pipette,  from  over  the 
surface  of  the  layer  of  magnesium  carbonate  and  trans- 
ferred to  a  second  flask,  and  similarly  after  four  or  five 
days  from  this  to  a  third  flask,  and  so  on.  As  this 
medium  does  not  offer  conditions  favorable  to  the 


532  BA  CTKRIOLOG  Y. 

growth  of  bacteria  requiring  organic  matter  for  their 
development,  those  that  Avere  originally  introduced  with 
the  soil  quickly  disappear,  and  ultimately  only  the  nitri- 
fying organisms  remain.  These  are  seen  as  an  almost 
transparent  film  attached  to  the  clumps  and  granules  of 
magnesium  carbonate  on  the  bottom  of  the  flask. 

For  their  cultivation  upon  a  solid  medium  Winograd- 
sky  employs  a  mineral  gelatin,  the  gelatinizing  principle 
of  which  is  silicic  acid.  A  solution  of  from  3  to  4  per 
cent,  of  silicic  acid  in  distilled  water,  and  having  a  spe- 
cific gravity  of  1.02,  remains  fluid  and  can  be  preserved 
in  flasks  in  this  condition.  (Kiihne.)  Gelatinization  oc- 
curs after  the  addition  of  certain  salts  to  such  a  solution, 
and  will  be  more  or  less  complete  according  to  the  pro- 
portion of  salts  added.  The  salts  that  have  given  the 
best  results  and  the  method  of  mixing  them  are  as  fol- 
lows : 

f  Ammonium  sulphate 0.4  gramme. 

a  <  Magnesium  sulphate 0.05  " 

( Calcium  chloride trace. 

f  Potassium  phosphate 0.1  gramme. 

6  <  Sodium  carbonate 0.6  to  0.9  " 

[  Distilled  water 100  c.c. 

The  sulphates  and  chloride  (a)  are  mixed  in  50  c.c. 
of  the  distilled  water,  and  the  phosphate  and  carbonate 
(b)  in  the  remaining  50  c.c.,  in  separate  flasks. 

Each  flask  is  then  sterilized  with  its  contents,  which 
after  cooling  are  mixed ;  the  mixture  representing  the 
solution  of  mineral  salts  is  to  be  added  to  the  silicic 
acid,  little  by  little,  until  the  proper  degree  of  consist- 
ency is  obtained  (that  of  ordinary  nutrient  gelatin). 
This  part  of  the  process  is  best  conducted  in  a  culture- 
dish.  If  it  is  desired  to  separate  the  colonies,  as  in  an 
ordinary  plate,  the  inoculation  and  mixing  of  the  mate- 


THE  NITRIFYING  BACTERIA.  533 

rial  introduced  must  be  done  before  gelatinization  is 
complete ;  if  the  material  is  to  be  distributed  over  only 
the  surface  of  the  medium,  then  the  mixture  must  first 
be  allowed  to  solidify. 

By  the  use  of  the  silicate-gelatin  Winogradsky  has 
isolated  from  the  gelatinous  film  in  the  bottom  of  fluids 
undergoing  nitrification  a  bacillus  which  he  believes  to  be 
associated  with  the  nitro-monas  in  the  nitrifying  process. 

Our  knowledge  of  these  organisms  is  as  yet  too  im- 
perfect to  permit  of  a  complete  description.  What  has 
been  said  will  serve  to  indicate  the  direction  in  which 
further  studies  of  the  subject  should  be  prosecuted. 
(For  further  details,  the  reader  is  referred  to  the  original 
contributions  and  to  current  literature  on  the  subject.1) 

In  addition  to  the  bacteria  concerned  in  decomposition 
and  nitrification  there  are  occasionally  present  in  the 
soil  micro-organisms  possessing  disease-producing  prop- 
erties. Conspicuous  among  these  may  be  mentioned 
the  bacillus  of  malignant  oedema  (vibrion  septique  of 
the  French),  the  bacillus  of  tetanus,  and  the  bacillus 
of  symptomatic  anthrax  (Ramchbrand  (Ger.)  ;  charbon 
xymptomatique  (Fr.)).  It  is  sometimes  due  to  the  pres- 
ence of  one  or  the  other  of  these  organisms  that  wounds 
to  which  soil  has  had  access  (crushed  wounds  from  the 
wheels  of  cars  or  wagons,  wounds  received  in  agricultural 
work,  etc.)  are  followed  by  such  grave  consequences. 

1  Winogradsky :  Annales  de  1'Institut  Pasteur,  1890,  tome  iv. ;  1891, 
tome  v. 

Jordan  and  Richards :  Report  of  State  Board  of  Health  of  Massa- 
chusetts, "  Purification  of  Sewage  and  Water,"  1890,  vol.  ii.  p.  864. 

Frankland,  G.  C.  and  P.  F.:  Proceedings  of  Royal  Society,  London, 
1N90,  xlvii. 

Wiuogradsky  and  Omeliansky :  "  Ueber  den  Einfluss  der  organisaten 
Substangen  auf  der  arbeitder  nitrifizierenden  Mikroben,"  Centralblatt 
fur  Bakteriologie,  1899,  Abt.  ii.  Bd.  v  S.  329. 


534  BACTERIOLOGY. 

BACILLUS   TETANI,  NICOLAIER,  1884. 

In  1884  Nicolaier  produced  tetanus  in  mice  and  rab- 
bits by  the  subcutaneous  inoculation  of  particles  of 
garden-earth,  and  demonstrated  that  the  pus  produced 
at  the  point  of  inoculation  was  capable  of  reproducing 
the  disease  in  other  mice  and  rabbits.  He  did  not  suc- 
ceed in  isolating  the  organism  in  pure  culture.  In  1884 
Garle  and  Rattone,  and  in  1886  Rosenbach,  demon- 
strated the  infectious  nature  of  tetanus  as  it  occurs  in 
man  by  producing  the  disease  in  animals  by  inoculating 
them  with  secretions  from  the  wounds  of  individuals 
aifected  with  the  disease.  In  1889  Kitasato  obtained 
the  bacillus  of  tetanus  in  pure  culture,  and  described 
his  method  of  obtaining  it  and  its  biological  peculiarities 
as  follows : 

METHOD  OF  OBTAINING  IT. — Inoculate  several  mice 
subcutaneously  with  secretions  from  the  wound  of  a 
case  of  typical  tetanus.  This  material  usually  contains 
not  only  tetanus  bacilli,  but  other  organisms  as  well,  so 
that  at  autopsy,  if  tetanus  results,  there  may  be  more  or 
less  suppuration  at  the  seat  of  inoculation  in  the  mice. 
In  order  to  separate  the  tetanus  bacillus  from  the  others 
that  are  present  the  pus  is  smeared  upon  the  surface 
of  several  slanted  blood-serum  or  agar-agar  tubes  and 
placed  at  37°  to  38°  C.  After  twenty-four  hours  all 
the  organisms  will  have  developed,  and  microscopic 
examination  will  usually  reveal  the  presence  of  a  few 
tetanus  bacilli,  recognizable  by  their  shape,  viz.,  that  of 
a  small  pin,  with  a  spore  representing  the  head.  After 
forty-eight  hours  at  38°  C.  the  culture  is  subjected  to  a 
temperature  of  80°  C.  in  a  water-bath  for  from  three- 
quarters  to  one  hour.  At  the  end  of  this  time  series  of 


BACILLUS  TETANI.  535 

plates  or  Esmarch  tubes  of  slightly  alkaline  gelatin  are 
made  with  very  small  amounts  of  the  culture  and  kept 
in  an  atmosphere  of  hydrogen  (see  page  220).  They 
are  then  kept  at  from  18°  to  20°  C.,  and  at  the  end 
of  about  a  week  the  tetanus  bacillus  begins  to  appear 
in  the  form  of  colonies.  After  about  ten  days  the 
colonies  should  not  only  be  examined  microscopically, 
but  each  colony  that  has  developed  in  the  hydrogen 
atmosphere  should  be  obtained  in  pure  culture  and 
again  grown  under  the  same  conditions.  The  colonies 
that  grow  only  without  oxygen,  and  which  are  com- 
posed of  the  pin-shaped  organisms,  must  be  tested  upon 
mice.  If  they  represent  growths  of  the  tetanus  bacillus, 
the  typical  clinical  manifestations  of  the  disease  will  be 
produced  in  these  animals. 

In  obtaining  the  organism  from  the  soil  much  diffi- 
culty is  experienced.  Here  are  encountered  a  number 
of  spore-bearing  organisms  that  are  facultative  in 
their  relation  to  oxygen,  and  are  therefore  very  difficult 
to  eliminate ;  and  there  is,  moreover,  one  in  particular 
that,  like  the  tetanus  bacillus,  forms  a  polar  spore. 
This  spore  is,  however,  less  round  and  much  more  oval 
than  that  of  the  tetanus  bacillus,  and  gives  to  the 
organism  containing  it  more  the  shape  of  a  javelin  (or 
clostridium,  properly  speaking)  than  that  of  a  pin,  the 
characteristic  shape  of  the  spore-bearing  tetanus  organ- 
ism. It  is  non-pathogenic,  and  grows  both  with  and 
without  oxygen,  and  should,  consequently,  not  be  mis- 
taken for  the  latter  bacillus.  It  must  also  be  borne  in 
mind  that  there  are  occasionally  present  in  the  soil  still 
other  bacilli  which  form  polar  spores,  and  which,  when 
in  this  stage,  are  almost  identical  in  appearance  with 
the  tetanus  bacillus ;  but  they  will  usually  be  found  to 


536 


BACTERIOLOGY. 


differ  from  it  in  their  relation  to  oxygen,  and  they  are 
also  without  disease-producing  properties. 

MORPHOLOGY. — In  the  vegetating  stage  it  is  a  slen- 
der rod  with  rounded  ends.  It  may  appear  as  single 
rods,  or,  in  cultures,  as  long  threads.  It  is  motile,  though 
not  actively  so.  The  motility  is  rendered  somewhat 
more  conspicuous  by  examining  the  organism  upon  a 

warm  stage. 

FIG.  86. 


Bacillus  tetani.   A.  Vegetative  stage.   B.  Spore-stage,  showing  pin-shapes. 


At  the  temperature  of  the  body  it  rapidly  forms 
spores.  These  are  round,  thicker  than  the  cell,  and 
usually  occupy  one  of  its  poles,  giving  to  the  rod  the 
appearance  of  a  small  pin.  (Fig.  86.)  When  in  the 
spore-stage  it  is  not  motile. 

It  is  stained  by  the  ordinary  aniline  stain  ing-reagents. 
It  retains  the  color  when  stained  by  Gram's  method. 

CULTURAL,  PECULIARITIES. — It  is  an  obligate  anae- 
robe, and  cannot  be  brought  to  development  under 
access  of  oxygen.  It  thrives  in  an  atmosphere  of 
pure  hydrogen,  but  not  in  one  of  carbonic  acid. 


B  A  C1L  L  US   TETA  NL 


537 


FIG.  87. 


It  grows  in  ordinary  nutrient  gelatin  and  agar-agar 
of  a  slightly  alkaline  reaction.  Gelatin  is  slowly  lique- 
fied, with  the  coincident  production  of  a  small  amount 
of  gas.  Neither  agar-agar  nor 
blood-serum  is  liquefied  by  its 
growth. 

The  addition  to  the  media  of 
from  1.5  to  2  per  cent,  of  glucose, 
0.1  per  cent,  of  indigo-sodium 
sulphate,  or  5  per  cent,  by  volume 
of  blue  litmus  tincture  favors  its 
growth. 

It  grows  well  in  alkaline  bouil- 
lon under  an  atmosphere  of  hy- 
drogen. 

Under  artificial  conditions  it 
may  be  cultivated  through  nu- 
merous generations  without  loss 
of  virulence. 

APPEARANCE  OF  THE  COLO- 
NIES.— Colonies  of  bacillus  tetani 
on  gelatin  under  an  atmosphere 
of  hydrogen  have,  in  their  early 
stages  somewhat  the  appearance 
of  the  colonies  of  the  common 
bacillus  subtilis  in  their  earliest 
stages,  viz.,  they  have  a  dense, 
felt-like  centre  surrounded  by  a 
fringe  of  delicate  radii.  The 
liquefaction  is  so  slow  that  the 
appearance  is  retained  for  a  rela- 
tively long  time,  but  eventually 
becomes  altered.  In  very  old 


Colonies  of  the  tetanus 
bacillus  four  days  old.made 
by  distributing  the  organ- 
isms through  a  tube  nearly 
filled  with  glucose-gel- 
atin. Cultivation  in  an  at- 
mosphere of  hy*drogen. 
(From  FRANK KL  nnd 

PFEIFFEB.) 


538  BACTERIOLOGY. 

colonies  the  entire  mass  is  made  up  of  a  number  of 
distinct  threads  that  give  it  the  appearance  of  a  com- 
mon mould.  (See  Fig.  87.) 

In  stab-cultures  made  in  tubes  about  three-quarters 
filled  with  gelatin  growth  begins  at  about  1.5  to  3  cm. 
below  the  surface,  and  gradually  assumes  the  appearance 
of  a  cloudy,  linear  mass,  with  prolongations  radiating 
into  the  gelatin  from  all  sides.  Liquefaction  with  coinci- 
dent gas-production  results,  and  may  reach  almost  to 
the  surface  of  the  gelatin. 

RELATION  TO  TEMPERATURE  AND  TO  CHEMICAL 
AGENTS. — It  grows  best  at  a  temperature  of  from 
36°  to  38°  C. ;  gelatin  cultures  kept  at  from  20°  to 
25°  C.  begin  to  grow  after  three  or  four  days.  In  an 
atmosphere  of  hydrogen  at  from  18°  to  20°  C.  growth 
does  not  usually  occur  before  one  week.  No  growth 
occurs  below  14°  C.  At  the  temperature  of  the  body 
spores  are  formed  in  cultures  in  about  thirty  hours, 
whereas  in  gelatin  cultures  at  from  20°  to  25°  C.  they 
do  not  usually  appear  before  a  week,  when  the  lower 
part  of  the  gelatin  is  quite  fluid. 

Spores  of  the  tetanus  bacillus  when  dried  upon  bits 
of  thread  over  sulphuric  acid  in  the  desiccator  and  sub- 
sequently kept  exposed  to  the  air,  retain  their  vitality 
and  virulence  for  a  number  of  months.  Their  vitality 
is  not  destroyed  by  an  exposure  of  one  hour  to  80°  C. ; 
on  the  other  hand,  an  exposure  of  five  minutes  to 
100°  C.  in  the  steam  sterilizer  kills  them.  They  resist 
the  action  of  5  per  cent,  carbolic  acid  for  ten  hours,  but 
succumb  when  exposed  to  it  for  fifteen  hours.  In  the 
same  solution,  plus  0.5  per  cent,  of  hydrochloric  acid, 
they  are  no  longer  active  after  two  hours.  They  are 
killed  when  acted  upon  for  three  hours  by  corrosive 


BACILLUS  TETANI.  539 

sublimate,  1  :  1000,  and  in  thirty  minutes  by  the  same 
solution  plus  0.5  per  cent,  of  hydrochloric  acid. 

ACTION  UPON  ANIMALS. — After  subcutaneous  inocu- 
lation of  mice  with  minute  portions  of  a  pure  culture 
of  this  organism  tetanus  develops  in  twenty-four  hours 
and  ends  fatally  in  from  two  to  three  days.  Rats, 
guinea-pigs,  and  rabbits  are  similarly  affected,  but  only 
by  larger  doses  than  are  required  for  mice,  the  fatal 
dose  for  a  rabbit  being  from  0.3  to  0.5  c.c.  of  a  well- 
developed  bouillon  culture.  The  period  of  incubation 
for  rats  and  guinea-pigs  is  twenty-four  to  thirty  hours, 
and  for  rabbits  from  two  to  three  days.  Pigeons  are 
but  slightly,  if  at  all,  susceptible. 

The  tetanic  convulsions  always  appear  first  in  the 
parts  nearest  the  seat  of  inoculation,  and  subsequently 
become  general. 

At  autopsies  upon  animals  that  have  succumbed  to 
inoculations  with  pure  cultures1  of  bacillus  tetani  there 
is  little  to  be  seen  by  either  macroscopic  or  micro- 
scopic examination,  and  cultures  from  the  site  of  inocu- 
lation are  often  negative  in  so  far  as  finding  the  tetanus 
bacillus  is  concerned.  At  the  site  of  inoculation  there  is 
usually  only  a  hypersemic  condition.  In  uncomplicated 
cases  there  is  no  suppuration.  The  internal  organs  do  not 
present  any  macroscopic  change,  and  culture-methods 
of  examination  show  them  to  be  free  from  bacteria. 
The  death  of  the  animal  results  from  the  absorption  of 
a  soluble  poison,  either  produced  by  the  bacteria  at  the 
site  of  inoculation  or,  which  seems  more  probable,  pro- 

1  Animals  and  human  beings  that  have  become  infected  with  this 
organism  in  the  ordinary  way  commonly  present  a  condition  of  sup- 
puration at  the  site  of  infection  ;  this  is  probably  not  due,  however,  to 
the  tetanus  bacillus,  but  to  other  bacteria  that  have  also  gained  access 
to  the  wound  at  the  time  of  infection. 


540  BA  CTERIOL  O  G  r. 

duced  by  the  bacteria  in  the  culture  from  which  they  are 
obtained  and  introduced  with  them  into  the  tissues  of 
the  animal  at  the  time  of  inoculation.  In  support 
of  the  latter  hypothesis  :  mice  have  been  inoculated  with 
pure  cultures  of  this  organism  ;  after  one  hour  the  point 
at  which  the  inoculation  was  made  was  excised  and  the 
tissues  cauterized  with  a  hot  iron ;  notwithstanding 
the  short  time  during  which  the  organisms  were  in 
contact  with  the  tissues  and  the  subsequent  radical 
treatment,  the  animals  died  after  the  usual  interval 
and  with  the  typical  symptoms  of  tetanus. 

The  poison  produced  by  the  tetanus  bacillus,  and 
to  which  the  symptoms  of  the  disease  are  due,  has 
been  isolated  and  subjected  to  detailed  study ;  some 
of  its  toxic  peculiarities,  as  given  by  Kitasato,  are  as 
follows  : 1 

"When  cultures  of  this  organism  are  robbed  of  their 
bacteria  by  filtration  through  porcelain  the  filtrate  con- 
tains the  soluble  poison,  and  is  capable,  when  injected 
into  animals,  of  causing  tetanus. 

"  Inoculations  of  other  animals  with  bits  of  the 
organs  of  the  animal  dead  from  the  action  of  the  teta- 
nus poison  produce  no  result ;  but  similar  inoculations 
with  the  blood  or  with  the  serous  exudate  from  the 
pleural  cavity  always  result  in  the  appearance  of  teta- 
nus. The  poison  is,  therefore,  largely  present  in  the 
circulating  fluids. 

"The  greatest  amount  of  poison  is  produced  by  culti- 
vation in  fresh  neutral  bouillon  of  a  very  slightly  alka- 
line reaction. 

"  The  activity  of  the  poison  is  destroyed  by  an  ex- 

1  Zeitschrift  fiir  Hygiene,  1891,  Bd.  x.  S.  267. 


BAC1LLVS  TETANL  541 

posure  of  one  and  one-half  hours  to  55°  C. ;  of  twenty 
minutes  to  60°  C. ;  and  of  five  minutes  to  65°  C. 

"By  drying  at  the  temperature  of  the  body  under 
access  of  air  the  poison  is  destroyed ;  but  by  drying  at 
the  ordinary  temperature  of  the  room,  or  at  this  tem- 
perature in  the  desiccator  over  sulphuric  acid,  it  is  not 
destroyed. 

"  Diffuse  daylight  diminishes  the  intensity  of  the 
poison.  Its  intensity  is  preserved  for  a  much  longer 
time  when  kept  in  the  dark. 

"  Direct  sunlight  robs  it  of  its  poisonous  properties 
in  from  fifteen  to  eighteen  hours. 

"  Its  activity  is  not  diminished  by  diluting  a  fixed 
amount  with  water  or  nutrient  bouillon. 

"  Mineral  acids  and  strong  alkalies  lessen  its  intensity ." 

The  chemical  nature  of  this  poison  is  not  positively 
known,  but  according  to  the  observations  of  Brieger 
and  Cohn l  its  designation  of  "  Toxalbumen  "  is  a  mis- 
nomer, for  its  reactions  do  not  warrant  its  classification 
with  the  albumins  in  the  sense  in  which  the  word  is 
commonly  used.  When  obtained  in  a  pure,  concentrated 
form,  its  toxic  properties  are  seen  to  be  altered  by  acids, 
by  alkalies,  by  sulphuretted  hydrogen,  and  by  tempera- 
tures above  70°  C.  Even  when  carefully  protected  from 
light,  moisture,  and  air,  it  gradually  becomes  diminished 
in  strength,  doubtless  due  to  the  formation  of  "  toxons  " 
and  "  toxoids,"  analogous  to  those  observed  by  Ehrlich  in 
deteriorating  diphtheria  toxin.  When  freshly  prepared 
by  the  methods  of  the  authors  just  cited,  its  potency  is 
almost  incredible,  0.00005  milligramme  being  sufficient  to 
cause  fatal  tetanus  in  a  mouse  weighing  fifteen  grammes. 

1  Zeitschrift  fur  Hygieue  uud  Infektiouskrankheiten,  1893,  Bd.  xv. 
S.I. 


542  BA  CTERIOLOG  Y. 


two  physiologically  distinct  intoxicating  compounds  : 
the  one,  a  solvent  of  erythrocytes  —  a  "  tetanolysin  "  ; 
the  other,  a  specific  irritant  which,  through  its  influence 
upon  the  central  nervous  system,2  accounts  for  the  phe- 
nomena by  which  tetanus  is  characterized  ;  to  this  latter 
the  designation  "  tetanospasmin  "  is  given.  Madsen's 
observations,  furthermore,  confirm  the  deductions  of 
Ehrlich  concerning  the  molecular  structure  of  bacterial 
toxins  in  general,  to  the  effect  that  the  molecule  of  tet- 
anolysin, like  that  of  diphtheria  toxin,  is  a  complex  of 
at  least  two  physiologically  unlike  groups  ;  the  one, 
characterized  by  its  marked  combining  tendencies  (for 
antitoxin),  the  so-called  haptophore  group  ;  the  other, 
distinguished  for  its  intoxicating  quality,  the  so-called 
toxophore  group. 

The  principles  involved  in  the  induction  of  the  anti- 
toxic state  against  diphtheria  are  likewise  applicable  to 
tetanus  ;  in  fact,  the  fundamental  observations  upon  the 
generation  of  antitoxin  in  the  living  animal  body  were 
made  in  the  course  of  studies  on  tetanus  ;  they  were 
subsequently  applied  to  the  study  of  diphtheria,  with 
the  results  already  noted.  It  is  needless  to  enter  here 
upon  the  details  essential  to  the  production  of  tetanus 
antitoxin  ;  to  all  intents  and  purposes,  they  are  identical 
with  those  given  in  the  section  on  diphtheria.  Briefly 
stated,  animals  may  be  rendered  immune  from  tetanus 
by  the  repeated  injection  of  gradually  increasing  non- 
fatal  doses  of  tetanus  toxin  ;  when  immunity  is  estab- 

1  Madsen  :  "  Ueber  Teanolysin,"  Zeitschrift  fur  Hygiene  und  Infek- 
tionskrankheiten,  1899,  Bd.  xxxii.  S.  214.. 

2,See  paper  by  Wassermann  and  Takaki  :  Berliner  klinische  Woch- 
enschrift,  1698,  No.  1,  S.  5. 


BACILLUS   TETANL  543 

lished,  the  circulating  blood  contains  a  body,  antitoxin, 
that  combines  directly  with  tetanus  toxin  in  a  test-tube, 
and  thereby  renders  it  physiologically  inactive  (non-in- 
toxicating) ;  and  the  serum  of  the  immune  animal  is 
not  only  capable  of  protecting  non-immune,  susceptible 
animals  from  the  poisonous  action  of  tetanus  toxin 
(within  limits),  but  also  against  the  effects  of  the  living 
tetanus  bacillus  as  well. 

Tetanus  antitoxin,  though  the  first  antitoxin  discov- 
ered and  frequently  employed  in  the  treatment  of 
tetanus,  has  not  yielded  as  brilliant  results  as  those 
obtained  with  diphtheria  antitoxin.  There  are  two 
important  reasons  why  tetanus  antitoxin  may  never  be 
expected  to  yield  such  satisfactory  results  as  does 
diphtheria  antitoxin.  First,  diphtheria  infection  can  be 
diagnosed  by  bacteriological  methods  and  the  antitoxin 
administered  long  before  any  very  marked  constitutional 
symptoms  have  developed,  and  consequently  long  before 
the  diphtheria  toxin  has  had  time  to  bring  about  very 
serious  tissue  alterations.  In  tetanus  it  is  impossible  to 
make  such  a  definite  bacteriological  examination,  and 
very  frequently  the  first  manifestation  of  the  disease  is 
the  twitching  of  the  muscles  which  is  the  antecedent 
sign  of  the  tetanic  convulsions.  When  these  clinical 
manifestations  have  developed  in  tetanus  there  is  already 
very  serious  involvement  of  the  central  nervous  system 
by  the  action  of  the  tetanus  toxin  upon  the  nerve  cells. 
The  second  reason  why  tetanus  antitoxin  is  likely  to 
prove  less  helpful  than  diphtheria  antitoxin  is  that  the 
tetanus  toxin  seems  to  have  very  great  affinity  for  the 
cells  of  the  central  nervous  system,  while  the  cells  and 
tissues  of  the  body  affected  primarily  by  diphtheria  toxin 
are  of  far  less  vital  importance. 


544  £.4  CTPIRIOLOG  Y. 

In  the  use  of  tetanus  antitoxin  it  is  advisable  to 
employ  it  as  early  as  possible  and  to  give  repeated  doses 
until  the  symptoms  are  relieved.  Whether  the  subdural 
administration  of  the  antitoxin  will  be  of  greater  value 
than  the  subcutaneous  administration  is  as  yet  unsolved. 

A  great  deal  of  benefit  is  also  likely  to  result  from 
the  administration  of  tetanus  antitoxin  as  a  prophy- 
lactic in  the  treatment  of  wounds  in  which  infection  by 
the  tetanus  bacillus  is  possible.  The  prophylactic  injec- 
tion of  the  tetanus  antitoxin  in  these  cases,  however, 
should  always  be  accompanied  by  the  most  rigid  aseptic 
and  antiseptic  treatment  of  the  wound,  and  under  these 
conditions  it  is  more  or  less  doubtful  which  of  these 
measures  is  of  the  greatest  value,  but  experience  seems 
to  indicate  that  the  antitoxin  has  a  distinct  prophylactic 
influence  in  these  cases. 

BACILLUS   CEDEMATIS,  LIBORIUS,  1886. 

The  bacillus  of  malignant  oedema,  also  known  as 
vibrion  septique,  is  another  pathogenic  form  almost 
everywhere  present  in  the  soil.  In  certain  respects  it 
is  a  little  like  bacterium  anthracis,  and  was  at  one 
time  confounded  with  it ;  but  it  diifers  in  the  marked 
peculiarity  of  being  a  strict  anaerobe.  It  was  first 
observed  by  Pasteur,  but  it  was  not  until  later  that 
Koch,  Laborious,  Kitt,  and  others  described  its  pecu- 
liarities in  detail.  It  can  often  be  obtained  by 
inserting  under  the  skin  of  rabbits  or  guinea-pigs  small 
portions  of  garden-earth,  street-dust,  or  decomposing 
organic  substances.  There  results  a  widespread  cedema, 
with  more  or  less  gas-production  in  the  tissues.  In  the 
cedematous  fluid  about  the  site  of  inoculation  the  organ- 
ism under  consideration  may  be  detected.  (Fig.  88,  A.) 


BACILLUS  CEDEMATIS. 


545 


It  is  a  rod  about  3  to  3.5  /j.  long  and  from  1  to  1.1  p. 
thick — i.  e.y  it  is  about  as  long  as  bacterium  anthracis, 
but  is  a  trifle  more  slender.  It  is  usually  found  in  pairs, 
joined  end  to  end,  but  may  occur  as  longer  threads; 
particularly  is  this  the  case  in  cultures.  When  in  pairs 

FIG.  88. 


Bacillus  cedematis.    A.  (Edema-fluid,  from  site  of  inoculation  of  guinea- 
pig,  showing  long  and  short  threads.    B.  Spore-formation,  from  culture. 

the  ends  that  approximate  are  squarely  cut,  while  the 
distal  extremities  are  rounded.  When  occurring  singly 
both  ends  are  rounded.  (How  does  it  differ  in  this 
respect  from  bacterium  anthracis  f)  It  is  slowly  motile, 
and  its  flagella  are  located  both  at  the  ends  and  along 

35 


546 


BACTERIOLOGY. 


FIG.  89. 


the  sides  of  the  rod.  It  forms  spores  that  are  usually 
located  in  or  near  the  middle  of  the  cells,  causing  fre- 
quently a  swelling  at  the  points  at  which  they  are 
located  and  giving  to  the  cell  a  more 
or  less  oval,  spindle,  or  lozenge  shape. 
(Fig.  88,  B.) 

It  is  an  obligate  anaerobe,  growing 
on  all  the  ordinary  media,  but  not  with 
access  of  oxygen.  It  grows  well  in 
an  atmosphere  of  hydrogen.  It  causes 
liquefaction  of  gelatin. 

In  tubes  containing  about  20  to  30  c.c. 
of  gelatin  that  has  been  liquefied,  inocu- 
lated with  a  small  amount  of  the  culture, 
and  then  rapidly  solidified  in  ice- water, 
growth  appears  in  the  form  of  isolated 
colonies  at  or  near  the  bottom  of  the 
tube  in  from  two  to  three  days  at 
20° 'C.  These  colonies,  when  of  from 
0.5  to  1  mm.  in  diameter,  appear  as 
spheres  filled  with  clear  liquid,  and 
are  difficult,  for  this  reason,  to  detect. 
(Fig.  89.)  As  they  gradually  increase 
in  size  the  contents  of  the  spheres  be- 
come cloudy  and  marked  by  fine  radi- 
ating stripes,  easily  to  be  detected  with 
the  aid  of  a  small  hand-lens.  In  deep 
of  malignant  stab-cultures  in  agar-agar  and  in  gela- 
thf  Tuiture.CeP(After  tin  development  occurs  only  along  the 
FRANKEL  and  PFEIF-  track  of  puncture,  at  a  distance  below 
the  surface.  Growth  is  frequently  ac- 
companied by  the  production  of  gas-bubbles. 

It  causes  rapid  liquefaction  of  blood-serum,  with  pro- 


Colonies  of  the  l>a- 


BACILLUS  (EDEMA TIS.  547 

duct-ion  of  gas-bubbles,  and  in  two  or  three  days  the 
entire  medium  may  have  become  converted  into  a 
yellowish,  semifluid  mass. 

The  most  satisfactory  results  in  the  study  of  the 
colonies  are  obtained  by  the  use  of  plates  of  nutrient 
agar-agar  kept  in  a  chamber  in  which  all  oxygen  has 
been  replaced  by  hydrogen.  The  colonies  appear  as  dull 
whitish  points,  irregular  in  outline,  and  when  viewed 
with  a  low-power  lens  are  seen  to  be  marked  by  a  net- 
work of  branching  and  interlacing  lines  that  radiate  in 
an  irregular  way  from  the  centre  toward  the  periphery. 

It  grows  well  at  the  ordinary  temperature  of  the 
room,  but  reaches  its  highest  development  at  the  tem- 
perature of  the  body. 

It  stains  readily  with  the  ordinary  aniline  dyes.  It 
does  not  stain  by  Gram's  method. 

PATHOGENESIS. — The  animals  known  to  be  suscepti- 
ble to  inoculation  with  this  organism  are  man,  horses, 
calves,  dogs,  goats,  sheep,  pigs,  chickens,  pigeons,  rab- 
bits, guinea-pigs,  and  mice.  Cases  are  recorded  in 
which  men  and  horses  have  developed  the  disease  after 
injuries,  doubtless  due  to  the  introduction  into  the 
wound,  at  the  time,  of  soil  or  dust  containing  the 
organism. 

If  one  introduce  into  a  pocket  beneath  the  skin  of  a 
susceptible  animal  about  as  much  garden-earth  as  can 
be  held  upon  the  point  of  a  penknife,  the  animal  fre- 
quently dies  in  from  twenty-four  to  forty-eight  hours. 
The  most  conspicuous  result  found  at  autopsy  is  a  wide- 
spread oedema  at  and  about  the  site  of  inoculation.  The 
oedcmatous  fluid  is  in  some  places  clear,  while  at  others  it 
may  be  stained  with  blood ;  it  is  usually  rich  in  bacilli 
(Fig.  88,  A)  and  contains  gas-bubbles.  Of  the  internal 


548  BA  CTERIOLOG  Y. 

organs  only  the  spleen  shows  much  damage.  It  is  large, 
dark  in  color,  and  contains  numerous  bacilli.  If  the 
autopsy  be  made  immediately  after  death,  bacilli  are 
rarely  found  in  the  blood  of  the  heart;  but  if  de- 
ferred for  several  hours,  the  organisms  will  be  found 
in  this  locality  also,  a  fact  that  speaks  for  their  multi- 
plication in  the  body  after  death.  At  the  moment 
of  death  they  are  present  in  varying  numbers  in  all 
the  internal  viscera  and  on  the  serous  surfaces  of  the 
organs. 

Of  all  animals  mice  are  probably  the  most  susceptible 
to  the  action  of  this  organism,  and  it  is  not  rare  to  find 
it  in  the  heart's  blood,  even  immediately  after  death. 
They  die,  as  a  result  of  these  inoculations,  in  from  six- 
teen to  twenty  hours. 

"When  a  pure  culture  is  used  for  inoculation  a  rela- 
tively large  amount  must  be  employed,  and  this  should  be 
introduced  into  a  deep  pocket  in  the  subcutaneous  tissues 
some  distance  from  the  surface.  In  continuing  the  in- 
oculations from  animal  to  animal  small  portions  of 
organs  or  a  few  drops  of  the  oedema-fluid  should  be 
used.  The  inoculation  may  also  be  successfully  made 
by  introducing  into  a  pocket  in  the  skin  bits  of  steril- 
ized thread  or  paper  upon  which  cultures  have  been 
dried. 

The  methods  for  obtaining  the  organism  in  pure  cult- 
ure, from  the  cadaver  of  an  animal  that  has  succumbed 
to  infection  by  the  bacillus  of  malignant  oedema,  are 
in  all  essential  respects  the  same  as  those  given 
for  obtaining  cultures  from  tissues  in  general ;  but  it 
must  be  remembered  that  the  organism  is  a  strict  anae- 
robe, and  will  not  grow  under  the  influence  of  oxygen. 
(See  methods  of  cultivating  anaerobic  species.) 


BACILLUS  CHAUVEI.  549 

In  certain  superficial  respects  this  bacillus  suggests, 
as  said  above,  bacterium  anthracis,  but  differs  from  it  in 
so  many  important  details  that  there  is  no  excuse  for 
confounding  the  two. 

NOTE. — From  what  has  been  said  of  this  organism, 
what  are  the  most  important  differential  points  between 
it  and  bacillus  anthracis  ?  Inoculate  several  mice  with 
small  portions  of  garden-earth  and  street-dust.  Isolate 
the  organism  that  agrees  most  nearly  with  the  descrip- 
tion here  given  for  the  bacillus  of  malignant  oedema. 
Compare  its  morphological,  biological,  and  pathogenic 
peculiarities  with  those  of  bacillus  anthracis  under  simi- 
lar circumstances ;  especially  its  action  on  animals  and 
its  appearance  in  the  tissues  and  fluids. 

Still  another  pathogenic  organism  that  may  be  present 
in  the  soil  is 

BACILLUS   CHAUVEI,    ARLOING,    CORNEVIN,    AND 
THOMAS,    1887. 

Synonyms :  The  bacillus  of  symptomatic  anthrax — Bacterie  du  char- 
bon  symptomatique  (Fr.) — Bacillus  des  rauschbrand  (Ger.). 

It  is  the  organism  concerned  in  the  production 
of  the  disease  of  young  cattle  and  sheep  commonly 
known  as  "  black  leg,"  "  quarter  evil/'  and  "  quarter 
ill,"  a  disease  that  prevails  in  certain  localities  dur- 
ing the  warm  months,  and  which  is  characterized  by 
a  peculiar  emphysematous  swelling  of  the  muscular 
and  subcutaneous  cellular  tissues  over  the  quarters. 
The  muscles  and  cellular  tissues  at  the  points  af- 
fected are  seen  on  section  to  be  saturated  with  bloody 
serum,  and  the  muscles  particularly  are  of  a  dark, 


550 


BACTERIOLOGY. 


almost  black  color.  In  these  areas,  in  the  bloody  trans- 
udates  of  the  serous  cavities,  in  the  bile,  and,  after 
death,  in  the  internal  organs,  the  organism  to  be  de- 
scribed can  always  be  detected.  It  is  manifest  from 
this  that  the  soil  of  localities  over  which  infected  herds 
are  grazing  may  readily  become  contaminated  through 
a  variety  of  channels,  and  thus  serve  as  a  source  of 
further  dissemination  of  the  disease. 

The  organism  was  first  observed  by  Feser,  and  subse- 
quently by  Bellinger  and  others.     The  most  complete 

FIG.  90. 


Bacillus  of  symptomatic  anthrax.    A.  Vegetative  stage— gelatin  culture. 
B.  Spore-forms—  agar-agar  culture. 

description  of  its  morphological  and  biological  peculi- 
arities is  that  of  Kitasato.1  The  following  is  from 
Kitasato' s  contributions:  it  is  an  actively  motile  rod 
about  3  to  5  /*  long  by  0.5  to  0.6  p  thick.  It  has 
rounded  ends,  and,  as  a  rule,  is  seen  singly,  though 
now  and  then  pairs  joined  end  to  end  may  occur.  It 

i  Kitasato :  Zeitschrift  fiir  Hygiene,  Bd.  vi.  S.  105;  Ed.  viii.  S.  55. 


BACILLUS  CHAUVEI. 


551 


has    no   tendency   to    form   very   long   threads.      (Fig. 
90,  A.) 

It  forms  spores,  and  when  in  this  stage  is  seen  to  be 
slightly  swollen  at  or  near  one   of  its 
poles,  the  location   in  which  the    spore  FlG-  9i. 

usually  appears.  (Fig.  90,  B.)  It  is 
markedly  prone  to  undergo  degenerative 
changes,  and  involution-forms  are  com- 
monly seen  not  only  in  fresh  cultures, 
but  in  the  tissues  of  affected  animals  as 
well. 

Though  actively  motile  when  in  the 
vegetative  stage,  it,  like  all  other  motile 
spore-forming  bacilli,  loses  this  property 
and  becomes  motionless  when  spores  are 
forming. 

It  is  strictly  anaerobic  and  cannot  be 
cultivated  in  an  atmosphere  in  which 
free  oxygen  is  present.  It  grows  best 
under  hydrogen,  and  does  not  grow  under 
carbonic  acid. 

The  media  most  favorable  to  its  growth 
are  those  containing  glucose  (1.5  to  2  per 
cent.),  glycerin  (4  to  5  per  cent.),  or  some 
other  reducing-body,  such  as  indigo- 
sodium  sulphate,  sodium  formate,  etc.  Coi0nies  of  the 

When  cultivated   upon  gelatin  plates    bacillus  of  symp- 

f>  ,       -,  !       tomatic     anthrax, 

in  an  atmosphere  ot  hydrogen  the  col-    in    deep    geiatin 
onios  appear  as  irregular,  slightly  lobu-    culture.       (After 

.          ,.  FRANKEL  and 

lated  masses.     After  a  short  time  lique-    PPEIFFEB.) 
faction  of  the  gelatin  occurs  and  the  col- 
ony presents  a  dark,  dense,  lobulated  and  broken  centre, 
surrounded  by  a  much  more  delicate,  fringe-like  zone. 


552  BACTERIOLOGY. 

When  distributed  through  a  deep  layer  of  liquefied 
gelatin  that  is  subsequently  solidified  colonies  develop 
at  only  the  lower  portions  of  the  tube.  The  single 
colonies  appear  as  discrete  globules  that  cause  rapid 
liquefaction  of  the  gelatin,  and  ultimately  coalesce 
into  irregular,  tabulated  liquid  areas.  In  some  of  the 
larger  colonies  an  ill-defined,  concentric  arrangement 
of  alternate  clear  and  cloudy  zones  can  be  made  out. 
(Fig.  91.) 

In  deep  stab-cultures  in  gelatin  growth  begins  after 
about  two  or  three  days  at  20°  to  25°  C.  It  begins 
usually  at  about  one  or  two  centimetres  below  the  sur- 
face, and  causes  slow  liquefaction  at  and  around  the 
track  of  its  development.  During  its  growth  gas- 
bubbles  are  produced. 

In  deep  stab-cultures  in  agar-agar  at  37°  to  38°  C. 
growth  begins  in  from  twenty-four  to  forty-eight  hours, 
also  at  about  one  or  two  centimetres  below  the  surface, 
and  is  accompanied  by  the  production  of  gas-bubbles. 
There  is  produced  at  the  same  time  a  peculiar,  pene- 
trating odor  somewhat  suggestive  of  that  of  rancid 
butter.  Under  these  conditions  spores  are  formed  after 
about  thirty  hours. 

It  grows  well  in  bouillon  of  very  slightly  acid  reac- 
tion under  hydrogen,  but  does  not  retain  its  virulence 
for  so  long  a  time  as  when  cultivated  upon  solid  media. 
In  this  medium  it  develops  in  the  form  of  white  flocculi 
that  sink  ultimately  to  the  bottom  of  the  glass  and  leave 
the  supernatant  fluid  quite  clear.  If  the  vessel  be  now 
gently  shaken,  these  delicate  flakes  are  distributed  homo- 
geneously through  it.  In  bouillon  cultures  there  is 
often  seen  a  delicate  ring  of  gas-bubbles  round  the 
point  of  contact  of  the  tube  and  the  surface  of  the 


EACfLLUS  CHAUVEL  553 

bouillon.  There  is  produced  also  a  peculiar,  penetrat- 
ing, sour  or  rancid  odor. 

It  grows  best  at  the  body-temperature — L  e.,  from  37° 
to  38°  C. — but  can  also  be  brought  to  development  at 
from  16°  to  18°  C.  Below  14°  C.  no  growth  is  seen. 
Spore-formation  appears  much  sooner  at  the  higher  than 
at  the  lower  temperatures.  When  its  spores  are  dried 
upon  bits  of  thread  in  the  dessiccator  over  sulphuric  acid, 
and  then  kept  under  ordinary  conditions,  they  retain 
their  vitality  and  virulence  for  many  months.  Sim- 
ilarly, bits  of  flesh  from  the  affected  areas  of  animals 
dead  of  this  disease,  when  completely  dried,  are  seen  to 
retain  for  a  long  time  the  power  of  reproducing  the 
disease.  The  spores  are  tolerably  resistant  to  the  in- 
fluence of  heat :  when  subjected  to  a  temperature  of 
80°  C.  for  one  hour  their  virulence  is  not  affected,  but 
an  exposure  to  100°  C.  for  five  minutes  destroys 
them.  They  are  also  seen  to  be  somewhat  resistant  to 
the  action  of  chemicals  :  when  exposed  to  5  per  cent, 
carbolic  acid  they  retain  their  disease-producing  prop- 
erties for  about  ten  hours,  whereas  the  vegetative  forms 
are  destroyed  in  from  three  to  five  minutes  ;  in  corro- 
sive sublimate  solution  of  the  strength  of  1  : 1000  the 
spores  are  killed  in  two  hours. 

When  gelatin  cultures  are  examined  microscopically 
the  organisms  are  usually  seen  as  single  rods  with 
rounded  ends.  When  cultivated  in  agar-agar  at  a 
higher  temperature  spores  are  formed  after  a  short 
time;  the  spores  are  oval,  slightly  flattened  on  their 
sides,  thicker  than  the  bacilli,  and,  as  stated,  fre- 
quently occupy  a  position  inclining  to  one  of  the  poles 
of  the  bacillus,  though  they  are  as  often  seen  in  the 
middle. 


554  B  A  CTERIOLOG  Y. 

Bacilli  containing  spores  are  usually  clubbed  or  spin- 
dle shape. 

This  bacillus  stains  readily  with  the  ordinary  aniline 
dyes.  It  is  decolorized  by  Gram's  method.  Its  spores 
may  be  stained  by  the  methods  usually  employed  in 
spore-staining. 

PATHOGENESIS. — When  susceptible  animals,  especi- 
ally guinea-pigs,  are  inoculated  in  the  deeper  subcutane- 
ous cellular  tissues  with  pure  cultures  of  this  organism, 
or  with  bits  of  tissue  from  the  affected  area  of  another 
animal  dead  of  the  disease,  death  ensues  in  from  one  to 
two  days.  It  is  preceded  by  rise  of  temperature,  loss 
of  appetite,  and  general  indisposition.  The  site  of 
inoculation  is  swollen  and  painful,  and  drops  of  bloody 
serum  may  sometimes  be  seen  exuding  from  it.  At 
autopsy  the  subcutaneous  cellular  tissues  and  under- 
lying muscles  present  a  condition  of  emphysema  and 
extreme  oedema.  The  redematous  fluid  is  often  blood- 
stained and  the  muscles  are  of  a  blackish  or  blackish - 
brown  color.  The  lymphatic  glands  are  markedly 
hypersemic.  The  internal  viscera  present  but  little 
alteration  visible  to  the  naked  eye.  In  the  blood- 
stained serous  fluid  about  the  point  of  inoculation  short 
bacilli  are  present  in  large  numbers.  These  often  pre- 
sent slight  swellings  at  the  middle  or  near  the  end. 
They  are  not  seen  as  threads,  but  lie  singly  in  the 
tissues.  Occasionally  two  will  be  seen  joined  end  to 
end.  If  the  autopsy  be  made  immediately  after  death, 
these  organisms  may  not  be  detected  in  the  internal 
organs ;  but  if  not  made  until  after  a  few  hours,  they 
will  be  found  there  also.  In  recent  autopsies  only  vege- 
tative forms  of  the  organism  may  be  found ;  but  later 
(in  from  twenty  to  twenty-four  hours)  spore-bearing  rods 


BACILLUS  CHAUVEL  555 

may  be  detected.  (How  does  this  compare  with  bacte- 
rium anthracis?)  By  successive  inoculations  of  suscepti- 
ble animals  with  serous  fluid  from  the  site  of  inoculation 
of  the  dead  animal  the  disease  may  be  reproduced. 

Cattle,  sheep,  goats,  guinea-pigs,  and  mice  are  sus- 
ceptible to  infection  with  this  organism,  and  present  the 
conditions  above  described ;  whereas  horses,  asses,  and 
white  rats  present  only  local  swelling  at  the  site  of  inoc- 
ulation. Swine,  dogs,  cats,  rabbits,  ducks,  chickens,  and 
pigeons  are,  as  a  rule,  naturally  immune  from  the  disease. 

Though  closely  simulating  the  bacillus  of  malignant 
oedema  in  many  of  its  peculiarities,  this  organism  can, 
nevertheless,  be  readily  distinguished  from  it.  It  is 
smaller ;  it  does  not  develop  into  long  threads  in  the 
tissues  ;  it  is  more  actively  motile,  and  forms  spores 
more  readily  in  the  tissues  of  the  animal  than  does  the 
bacillus  of  malignant  oedema.  In  their  relation  to  ani- 
mals they  also  differ;  for  instance,  cattle,  while  con- 
spicuously susceptible  to  symptomatic  anthrax,  are  prac- 
tically immune  from  malignant  oedema  ;  and  while  swine, 
dogs,  rabbits,  chickens,  and  pigeons  are  readily  infected 
with  malignant  oedema,  they  are  not,  as  a  rule,  suscepti- 
ble to  symptomatic  anthrax  Horses  are  affected  only 
locally,  and  not  seriously,  by  the  bacillus  of  symptomatic 
anthrax  ;  but  they  are  conspicuously  susceptible  to  both 
artificial  inoculation  and  natural  infection  by  the  bacil- 
lus of  malignant  oedema. 

The  distribution  of  the  two  organisms  over  the  earth's 
surface  is  also  quite  different.  The  oedema  bacillus  is 
present  in  almost  all  soils,  while  the  bacillus  of  symp- 
tomatic anthrax  appears  to  be  confined  to  certain  locali- 
ties, especially  places  over  which  infected  herds  have 
been  pastured. 


556  BACTERIOLOG  Y. 

A  single  attack  of  symptomatic  anthrax,  if  not  fatal, 
affords  subsequent  protection ;  while  infection  with  the 
malignant  redema  bacillus  appears  to  predispose  to  re- 
currence of  the  disease.  (Baumgarten.) 

BACTERIUM    WELCHII,    MIGULA,  1900. 

Synonym :  Bacillus  aerogenes  capsulatus,  Welch  and  Nuttall,  189:2. 

This  organism  consists  of  straight  or  slightly  curved 
rods  with  rounded  ends,  somewhat  thicker  than  bacte- 
rium anthracis,  varying  in  length  ranging  from  3  to  6 
microns ;  sometimes  longer  chains  or  threads  are  seen. 
The  rods  are  surrounded  by  a  transparent  capsule, 
whether  grown  in  artificial  media  or  obtained  from 
animal  bodies.  It  is  a  non-motile,  spore-forming 
organism,  and  is  strictly  anaerobic  in  character.  It 
stains  with  the  ordinary  aniline  dyes  and  by  the  Gram 
method. 

Under  anaerobic  conditions  the  organism  grows  on 
the  usual  culture  media  at  room  temperature,  and  forms 
large  quantities  of  gas  in  media  containing  carbohy- 
drates. Gelatin  is  not  liquefied.  In  agar-agar  the  col- 
onies are  usually  from  1  to  2  millimetres  in  diameter, 
but  may  be  as  large  as  1  centimetre  in  diameter.  They 
have  a  grayish-white  color,  are  flat,  round  or  irregular 
masses,  with  small  hair-like  projections  from  the  mar- 
gin. In  bouillon  there  is  a  diffuse  clouding  and  marked 
white  sediment.  Milk  is  quickly  coagulated.  On 
potato  there  is  a  grayish-white  layer. 

The  organism  grows  more  rapidly  at  30°  to  37°  C. 
than  at  18°  to  20°  C.  Cultures  on  agar-agar  and  bouil- 
lon have  a  slight  odor  resembling  old  lime.  Bouillon 
cultures  are  killed  after  ten  minutes  at  58°  C. 

Bacterium  Welch ii  was  first  described  bv  Welch  in 


BACTERIUM   WELCHIL  557 

1891,  and  subsequently  by  Welch  and  Nuttall *  in  the 
blood  and  internal  organs  of  a  patient  with  thoracic 
aneurism  opening  externally.  Autopsy  was  made  eight 
hours  after  death  and  the  vessels  were  found  to  contain 
large  numbers  of  gas  bubbles. 

Injections  of  considerable  quantities  of  cultures  into 
the  circulation  of  rabbits  did  not  kill  the  animals,  but 
if  the  animals  were  killed  after  being  inoculated  and 
were  then  allowed  to  lie  at  room  temperature  for  twenty- 
four  hours  the  organs  and  tissues  were  filled  with  gas 
bubbles. 

'Welch,  Howard,  Hitschman  and  Lilienthal,  Hirsch- 
berg,  and  others  have  shown  that  the  organism  is  fre- 
quently present  in  the  fa?ces  of  man  and  animals,  as 
well  as  in  the  soil  and  in  dust.  Schattenfroh  and  Grass- 
berger  also  found  the  organism  in  market  milk. 

BACILLUS  SPOROGENES  (KLEIN),  MIGULA,  1900. 
Synonym  :  Bacillus  enteritidis  sporogenes,  Klein,  1895. 

Klein  found  this  organism  in  the  intestinal  discharges 
of  infants  and  believed  it  had  some  relation  to  the  acute 
inflammatory  conditions  of  the  intestinal  tract  of  bottle- 
fed  infants.  The  organism  is  very  generally  distributed 
in  nature  and  can  be  very  readily  isolated  from  sewage 
by  appropriate  methods.  It  is  an  anaerobic,  spore-form- 
ing organism,  0.8  micron  in  width,  and  1.6  to  4.8  microns 
in  length.  It  is  actively  motile  and  flagella  have  been 
demonstrated. 

In  culture  media  containing  carbohydrates  this  organ- 
ism produces  gas  in  large  quantities.  Russell  analyzed 
the  gas  and  found  it  to  be  composed  principally  of 
methane.  Milk  and  other  sugar  media  in  which  the 

1  Welch  and  Nuttall:  Bulletin  Johns  Hopkins  Hospital,  No. 24, 1892. 


558  BA  CTERIOLOG  Y. 

organism  has  been  grown  have  a  distinct  odor  of  butyric 
acid. 

When  injected  subcutaneously  into  guinea-pigs  this 
organism  causes  most  marked  alterations.  There  is 
intense  inflammation  at  the  point  of  injection  with 
oedema  and  necrosis  and  the  surrounding  tissues  are 
filled  with  gas.  The  bacteria  are  distributed  throughout 
the  body  of  the  animal  and  can  be  isolated  in  pure  cul- 
ture from  the  blood  of  the  heart.  All  the  internal 
organs  are  intensely  congested. 


CHAPTER    XXV. 

Infection  and  immunity — The  types  of  infection  ;  intimate  nature  of 
infection — Septicaemia,  toxaemia,  variations  in  infectious  processes 
— Immunity,  natural  and  acquired,  active  and  passive — The 
hypotheses  that  have  been  advanced  in  explanation  of  immunity 
— Conclusions. 

AN  organism  capable  of  producing  disease  we  call 
pathogenic  or  infective,  and  the  process  by  which  it  pro- 
duces disease  we  know  as  infection.  Diseases,  therefore, 
that  depend  for  their  existence  upon  the  presence  of 
bacteria  in  the  tissues  are  infectious  diseases. 

What  is  the  mechanism  of  this  process  we  call  infec- 
tion ?  Is  it  due  to  the  mechanical  presence  of  living 
bacteria  in  the  body,  or  does  it  result  from  the  deposition 
in  the  tissues  of  substances  produced  by  these  bacteria 
that  are  either  locally  or  generally  incompatible  with 
life  ?  Or,  is  the  group  of  pathological  alterations  and 
constitutional  symptoms  seen  in  these  diseases  the  result 
of  abstraction  from  the  tissues,  by  the  bacteria  growing 
in  them,  of  substances  essential  to  the  vitality  of  both 
bacteria  and  tissues  ?  These  are  some  of  the  more 
important  questions  that  present  themselves  in  the 
course  of  analysis  of  this  interesting  phenomenon. 

Let  us  look  into  several  typical  infectious  diseases, 
note  what  we  find,  and  see  to  what  extent  the  observa- 
tions thus  made  will  aid  us  in  formulating  an  opinion. 
We  begin  with  a  study  of  those  diseases  in  which  there 
is  a  general  infection — i.  e.,  in  which  there  is  a  general  dis- 
tribution of  the  infective  agents  throughout  the  body. 

559 


560  BA  CTERIOL  0  G  Y. 

This  group  comprises  the  "  septicaemias/7  and  of  them 
the  disease  of  animals  known  as  anthrax  represents  a 
type  of  the  condition.  If  the  cadaver  of  an  animal 
dead  of  anthrax  be  examined  by  bacteriological  methods, 
there  will  be  discovered  present  in  all  the  organs 
and  tissues  an  organism,  a  bacterium,  of  definite  form 
and  biological  characteristics ;  and  if  the  organs,  and 
tissues  generally,  be  subjected  to  microscopic  examina- 
tion this  same  organism  will  be  found  and  always 
located  within  the  capillaries.  At  many  points  it  will 
be  seen  crowded  in  the  capillaries  in  such  numbers  as 
almost,  if  not  quite,  to  burst  them,  and  very  commonly 
their  lumen  for  a  considerable  extent  is  entirely  occluded 
by  the  growing  bacteria.  In  such  a  case  as  this  we  might 
be  tempted  to  conclude  that  death  had  resulted  from 
mechanical  interference  with  the  capillary  circulation. 
Suppose,  however,  we  subject  the  cultures  obtained  from 
this  animal  to  conditions,  either  chemical  or  thermal, 
that  are  not  particularly  favorable  to  their  normal  devel- 
opment, and  from  time  to  time  inoculate  susceptible 
animals  with  the  cultures  so  treated.  The  result  will  be 
that,  as  we  continue  to  expose  our  cultures  to  unfavorable 
surroundings,  the  period  of  time  that  is  required  for 
them  to  cause  the  death  of  animals  will,  in  some  cases, 
gradually  become  extended,  until  finally  death  will  not 
ensue  at  all  after  inoculation.  If,  as  these  animals  die, 
a  careful  record  of  the  condition's  found  at  autopsy  be 
kept  and  compared,  it  will  ultimately  be  noticed  that 
the  animals  that  die  at  a  longer  time  after  inoculation 
present  conditions  more  or  less  at  variance  with  those 
seen  in  the  original  animal  that  died  more  quickly  after 
having  been  inoculated  with  the  normal  organism.  These 
differences  usually  consist  in  a  diminution  of  the  num- 


INFECTION  AND  IMMUNITY.  561 

her  of  bacteria  that  appear  upon  culture  plates  from  the 
blood  and  internal  organs,  and  in  a  lessening  in  the 
amount  of  mechanical  obstruction  offered  to  the  circu- 
lation through  plugging  of  the  capillaries  by  masses  of 
bacteria,  as  detected  by  microscopic  examination  of  sec- 
tions of  the  organs ;  indeed,  this  latter  condition  may 
often  have  almost,  if  not  quite,  disappeared.  We  see  here 
an  animal  dead  from  the  invasion  of  the  same  organism 
that  produced  death  in  the  first  animal,  but  with  little 
or  none  of  those  particular  lesions  to  which  we  were 
inclined  to  attribute  the  death  of  that  animal.  It  is 
apparent,  then,  that  this  organism  with  which  we  have 
been  working  can  destroy  the  vitality  of  an  animal  in 
a  way  other  than  by  mechanically  obstructing  its  blood- 
vessels ;  it  possesses  some  other  means  of  destroying 
life.  Possibly  its  growth  in  the  tissues  is  accompanied 
by  the  production  of  soluble  poisons,  which  when  pres- 
ent in  the  blood  are  not  compatible  with  life. 

Let  us  see  if  the  study  of  another  group  of  infections 
will  furnish  any  evidence  in  support  of  such  an  hypoth- 
esis. Introduce  into  the  subcutaneous  tissues  of  a 
guinea-pig  a  small  amount  of  a  pure  culture  of  the  bacil- 
lus of  diphtheria.  In  three  or  four  days  the  animal 
dies.  We  proceed  with  our  autopsy  in  exactly  the  same 
way  that  we  did  with  the  animals  dead  of  anthrax, 
and  are  astonished  to  find  that  the  organs,  blood,  and 
tissues  generally  are  sterile,1  in  so  far  as  the  presence  of 
the  organism  with  which  the  animal  was  inoculated  is 
concerned,  and  by  both  cultural  and  microscopic  methods 
it  is  possible  to  detect  them  only  at  the  site  of  inocula- 
tion— i.  e.y  where  they  were  deposited.  It  is  very  evident 

1  In  by  far  fche  greater  number  of  cases  this  is  true ;  but  there  are 
isolated  exceptions  to  it. 


562  B  A  CTERIOLOG  Y. 

that  we  have  here  a  condition  with  which  mechanical 
plugging  of  the  capillaries  could  have  had  no  connec- 
tion, for  there  are  no  organisms  in  the  blood  to  interfere 
with  its  circulation.  Our  hypothesis  then  with  regard 
to  the  condition  found  in  our  first  case  of  anthrax  is 
again  of  doubtful  value.  Similarly,  if  an  animal  that  has 
died  of  tetanus  be  examined,  we  do  not  find  the  bacilli 
in  the  tissues  and  circulating  fluids  generally,  and,  in- 
deed, often  fail  to  find  them  even  at  the  point  of  injury. 
Plainly,  the  fatal  results  following  upon  inoculations 
with  the  diphtheria  and  the  tetanus  bacillus,  with  their 
accompanying  tissue-changes,  occur  from  the  presence 
of  a  something  that  cannot  be  detected  by  either  cult- 
ural or  microscopic  methods,  and  this  something  can  be 
only  a  soluble  substance  that  is  produced  by  the  growing 
bacteria  at  the  site  of  inoculation,  gains  access  to  the 
circulation,  and  through  this  channel  causes  death,  for 
it  is  scarcely  to  be  imagined  that  the  insignificant  wound 
made  in  the  course  of  inoculation  could  per  se  have  had 
this  effect.  In  other  words,  these  latter  animals  have 
died  from  what  is  called  toxaemia  (poison  in  the  blood), 
a  condition  distinctly  different  from  septicaemia,  as  seen 
in  our  first  animal  dead  of  anthrax. 

There  are,  again,  other  infectious  diseases,  many  of 
which  are  known  to  present  variations  from  what  might 
be  considered  a  typical  course,  that  may  still  further 
serve  to  support  the  view  that  infection  is  a  process  in 
which  the  mechanical  effect  of  organisms  in  the  circu- 
lating fluids  is  of  little  consequence.  Conspicuous 
among  these  are  the  infections  that  follow  upon  the 
introduction  into  the  tissues  of  susceptible  animals  of 
cultures  of  micrococcus  lanceolatus  (pneumococcus),  of 
the  bacillus  of  chicken  cholera,  and  of  the  organisms  con- 


INFECTION  AND  IMMUNITY.  563 

cerned  in  the  production  of  the  so-called  "  hemorrhagic 
septicaemias."  When  running  their  normal  course  the 
vpecific  organisms  of  these  diseases  cause  typical  septi- 
caemias in  susceptible  animals  ;  but  often,  from  causes  not 
entirely  clear,  the  animals  die  with  only  local  lesions,  or 
with  but  very  few  organisms  in  the  internal  viscera.  We 
see  here  conditions  analogous  to  those  observed  in  the 
two  experiments  with  anthrax,  viz.,  we  find  a  group  of 
diseases  that  are  properly  classed  as  septicaemias,  be- 
cause of  the  usual  general  invasion  of  the  body  by  the 
organisms  concerned  in  their  production,  but  which 
frequently  assume  a  purely  local  character — in  both 
instances  proving  fatal  to  the  animal  infected.  From 
what  we  have  seen  it  is  manifestly  probable  that, 
whether  these  diseases  be  designated  as  septicaemias  or 
as  toxaemias,  death  is  produced  in  all  instances  by 
poisonous  substances  that  are  generated  by  the  infecting 
bacteria.  In  the  case  of  typical  anthrax,  and  other 
varieties  of  septicaemia,  the  production  of  this  poison 
is  associated  with  the  general  dissemination  of  the 
organisms  throughout  the  body ;  while  in  those  infec- 
tions often  referred  to  as  toxaemias,  of  which  diphtheria 
may  be  taken  as  a  type,  the  poison  is  produced  by  the 
organisms  that  remain  localized  at  the  site  of  invasion, 
and  is  thence  disseminated  throughout  the  body  by  the 
circulating  fluids.  Infection  thus  far,  then,  appears  to 
be  a  chemical  process. 

In  still  another  group  of  infections  there  is  neither 
a  general  distribution  of  the  organisms  throughout  the 
vascular  system  nor  an  elaboration  of  toxins  that  can 
be  readily  separated  from  the  organisms  manufactur- 
ing them.  In  these  infections,  of  which  typhoid  fever 
and  Asiatic  cholera  may  be  taken  as  conspicuous  ex- 


564  BA  CTER IOLOG  Y. 

amples,  the  toxicity  of  the  invading  bacteria  appar- 
ently depends  upon  the  existence  of  intracellular  sub- 
stances of  a  poisonous  nature  that  have  thus  far  eluded 
all  efforts  to  satisfactorily  separate  them  from  the 
bodies  of  the  bacteria  in  which  they  develop.  The 
mechanism  by  which  this  group  of  bacteria  acts  is  as  yet 
far  from  clear.  We  only  know  that  the  presence  of 
members  of  this  group  in  the  bodies  of  susceptible  ani- 
mals is  accompanied  by  the  death  of  the  tissues  in  which 
they  are  located.  Whether  the  poisons  are  eliminated 
by  the  bacteria  as  secretions,  or  whether  they  are  set  free 
through  the  disintegration  of  bacteria  in  the  tissues  can- 
not be  said.  The  main  point  is,  however,  that,  both 
clinically  and  anatomically,  diseases  due  to  this  group 
of  bacteria  are  characterized  by  marked  evidence  of 
intoxication. 

BACTERIAL  TOXINS. — Through  special  investigations 
that  have  been  made  upon  the  products  of  growth  of 
certain  pathogenic  bacteria  the  opinion  that  infection  is 
a  chemical  process  receives  further  confirmation.  It 
has  been  found  possible  by  the  use  of  appropriate  methods 
to  isolate  from  among  the  mass  of  material  in  which 
certain  of  these  organisms  have  been  artificially  culti- 
vated substances  which,  when  separated  from  the 
bacteria  by  which  they  were  produced,  possess  the 
power  of  causing  in  animals  all  the  constitutional 
symptoms  and  pathological  tissue-changes  that  occur 
in  the  course  of  infection  by  the  organisms  themselves. 
In  some  instances  these  poisons— toxins,1  as  they  are 

1  The  term  "toxins  "  is  commonly  applied  to  amorphous,  nitrogenous 
poisons  produced  by  bacteria  in  both  living  tissues  ami  dead  substances ; 
while,  on  the  other  hand,  the  term  "  ptomains  "  relates  to  crystallizable, 
nitrogenous  poisons  that  are  formed  in  dead  tissue,  and  "  leucomains  " 
to  poisonous  and  non-poisonous  alkaloidal  bodies  that  occur  in  living 
tissues  as  a  result  of  physiological  metabolism. 


INFECTION  AND  IMMUNITY.  565 

collectively  called — appear  to  be  the  direct  result  of 
metabolic  changes  brought  about  by  bacteria  in  the 
medium  or  tissues  in  which  they  may  be  developing — 
i.  e.,  they  are  products  of  nutrition  that  pass  readily  into 
solution,  as  is  conspicuously  seen  in  the  case  of  the 
bacillus  of  diphtheria  and  of  tetanus  when  under  both 
artificial  cultivation  and  in  the  animal  body.  On  the 
other  hand,  as  said  above,  certain  bacteria  do  not  possess 
the  power  of  generating  or  secreting  such  poisons ;  they 
have,  nevertheless,  intimately  associated  with  their  pro- 
toplasmic bodies  poisonous  substances  that  manifest 
themselves  only  when  these  organisms  gain  access  to 
living  susceptible  tissues ;  thus  the  toxins  of  bacterium 
tuberculosis  and  of  microspira  comma  are  much  more 
conspicuously  present  in  the  protoplasm  of  these  bacteria 
than  in  the  fluids  in  which  they  have  grown. 

Buchner  has  isolated  from  several  species  of  bacteria 
"  bacterioproteins "  having  the  common  properties  of 
solubility  in  alkalies,  resistance  to  the  boiling  tempera- 
ture, attraction  of  leucocytes  (positive  chemotaxis T),  and 
pyogenic  powers. 

There  is  as  yet  little  agreement  of  opinion  as  to  the 
chemical  nature  of  toxins ;  but  it  is  probable  that  the 
group  comprises  diiferent  bodies  of  the  nature  of  globu- 
lins, nucleo-albumins,  peptones,  albumoses,  and  enzymes 
or  ferments. 

Toxic  ptomains  are  probably  not  conspicuously  con- 
cerned in  producing  the  characteristic  symptoms  of 
infection,  as  they  are  absent  from  cultures  of  certain 
highly  pathogenic  bacteria. 

In  particular  instances  the  production  of  poisonous 
principles,  even  under  artificial  conditions  of  cultivation, 
1  See  Chemotaxis. 


566  BACTERIOLOGY. 

is  most  astonishing,  and  poisons  are  generated  that  in  the 
degree  of  their  toxicity  exceed  anything  hitherto  known 
to  us.  For  instance,  the  potencies  of  the  poisons  that 
have  been  isolated  from  cultures  of  bacterium  diphtlierice 
and  of  bacillus  tetani  have  been  carefully  determined 
by  experiments  upon  animals,  and  it  has  been  found 
that  0.4  milligramme  of  the  -former  is  capable  of 
killing  eight  guinea-pigs,  each  weighing  400  grammes, 
or  two  rabbits,  each  weighing  3  kilogrammes  (Roux  and 
Yersin1);  and  that  0.0001  milligramme  of  the  latter 
will  produce  tetanus  in  a  mouse,  with  all  the  character- 
istic manifestations  of  the  disease  (Brieger  and  Cohn 2).3 
TOXOIDS  AND  TOXONES. — Ehrlich  conceives  the  toxin 
molecule  to  possess  two  atom-groups,  a  haptophore  group, 
by  means  of  which  it  unites  with  certain  cells  of  the  body, 
or  with  antitoxin ;  and  a  toxophore  group,  by  means  of 
which  it  produces  its  toxic  effects.  When  preserved  for  a 
time,  or  under  the  influence  of  various  chemical  and  phys- 
ical agents,  the  toxophore  group  of  the  toxin  molecule  de- 
teriorates, so  that  it  is  no  longer  capable  of  exerting  any 
toxic  action.  The  haptophore  group  is  less  easily  dis- 
turbed, and  hence  the  combining  power  of  the  toxin 
molecule  may  be  unaltered,  though  it  may  have  lost  its 
toxic  properties.  In  this  condition  it  is  spoken  of  as 
toxoid  or  toxone.  A  toxoid  or  toxone  is  still  capable 
of  inducing  antitoxin  formation  when  injected  into  an 
animal,  and  it  is  also  still  capable  of  neutralizing  anti- 
toxin in  the  same  proportions  as  before  the  alteration 
had  taken  place. 

1  Annales  de  1'Institut  Pasteur,  1889,  tome  iii.  p.  287. 

2  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  1893,  Bd.  xv. 
Heft  1. 

3  By  the  use  of  more  recently  devised  methods  we  are  enabled  to 
increase  still  further  the  toxicity  of  these  poisons;  especially  is  this 
the  case  with  regard  to  the  diphtheria  toxin. 


INFECTION  AND  IMMUNITY.  567 

MODE  OF  ACTION  OF  PATHOGENIC  BACTERIA. — 
The  development  of  our  knowledge  of  immunity  began 
with  the  recognition  of  the  relation  existing  between  the 
toxins  and  antitoxins.  It  was  found  that  on  the  injection 
into  animals  of  a  dose  of  toxin  certain  reactions  occurred 
varying  with  the  size  of  the  dose — that  is,  if  the  dose 
was  a  fatal  one  the  animal  died  within  a  definite  period 
of  time,  which  one  may  call  the  incubation  period.  This 
incubation  period  is  shorter  the  greater  the  amount  of 
toxin  injected.  If  the  amount  of  toxin  injected  is  less 
than  the  minimum  fatal  dose  the  animal  shows  after  a 
time  certain  reactions,  and  after  recovery  from  the 
effects  of  the  injection  larger  doses  of  the  toxin  can  be 
injected  without  destroying  the  animal.  At  the  same 
time  there  appears  in  the  serum  an  antibody  which  when 
injected  into  a  normal  animal  will  protect  it  against  the 
minimum  fatal  dose  of  the  toxin.  Of  great  importance 
in  the  relation  between  the  toxin  and  antitoxin  is  the  law 
of  multiple  proportions,  whereby  one  understands  a 
definite  relation  between  the  two — that  is,  a  definite 
amount  of  antitoxin  will  protect  a  normal  animal  against 
a  definite  amount  of  toxin.  A  multiple  dose  of  the 
serum  will  protect  against  multiple  doses  of  the  toxin 
of  the  same  proportion. 

FORMATION  OF  TOXIN  BY  BACTERIA. — The  num- 
ber of  bacteria  which  are  capable  of  producing  a  true 
toxin  is  quite  small.  Of  those  that  are  concerned  in 
human  pathology,  diphtheria  and  tetanus  organisms  are 
the  most  important.  For  the  majority  of  the  other  bac- 
teria toxin  formation  has  not  been  definitely  demon- 
strated. In  most  of  the  other  pathogenic  bacteria  it 
has  been  ascertained  that  the  toxic  action  which  they 
bring  about  rests  upon  the  poisonous  character  of  the 


568  BACTERIOLOGY. 

bacterial  protoplasm,  and  we  now  regard  the  toxic  action 
of  these  bacteria  to  be  due  to  the  formation  of  endotoxins 
or  intracellular  toxins. 

THE  ENDOTOXINS. — In  contradistinction  to  the  re- 
sults obtained  in  the  injection  of  the  soluble  toxins  the 
action  of  the  endotoxins  is  not  nearly  as  well  understood. 
The  injection  of  the  endotoxins  does  not  bring  about  the 
formation  of  anti-endotoxic  substances  in  the  serum. 
The  serum  of  animals  treated  with  the  endotoxins  has 
merely  a  bactericidal  action,  and  through  the  solution  of 
the  bacteria  protection  against  infection  is  seen,  but  mul- 
tiple doses  of  serum  do  not  act  against  corresponding 
multiple  doses  of  endotoxin.  The  treatment  of  animals 
with  endotoxin  does  not  protect  the  animal  body  against 
the  action  of  the  endotoxins. 

The  endotoxins  are  not  secreted  or  excreted  in  the 
culture  media  in  which  the  bacteria  are  cultivated,  but 
are  associated  with  the  bacterial  cells,  and  are  only  lib- 
erated through  the  solution  of  the  bacteria — that  is, 
through  bacteriolysis. 

Liberation  of  Endotoxins. — Bacteriolysis  may  occur, 
on  the  one  hand,  as  the  result  of  the  specific  reac- 
tion consisting  in  the  anchoring  of  the  amboceptors 
contained  in  an  immune  serum  through  the  receptors  of 
the  particular  organisms.  After  the  addition  of  comple- 
ment, which  is  normally  present  in  the  body,  union  takes 
place  with  the  receptor-amboceptor  group  and  bacterio- 
lysis follows. 

Besides  this  specific  bacteriolysis  there  is  also  a  non- 
specific bacteriolysis  in  which  the  endotoxins  contained  in 
the  bodies  of  bacteria  are  liberated — namely,  through 
autolytic  actions.  In  old  cultures  of  bacteria  one  often 
finds  only  few  individuals  and  these  are  usually  in  various 


INFECTION  AND  IMMUNITY.  569 

stages  of  degeneration.  The  different  species  of  bacte- 
ria vary  greatly  with  regard  to  their  proneness  to  auto- 
lysis.  In  some  of  them — for  instance,  microspira  comma, 
bacillus  typhosus,  etc. — the  autolysis  occurs  quite  early. 
The  poisonous  substances  contained  in  the  bodies  of  the 
organisms  thereby  pass  into  solution.  One  finds  in  fil- 
trates of  relatively  young  cultures  some  poisonous  action, 
which  is  still  greater  in  filtrates  from  old  cultures.  Other 
bacteria,  as  for  instance,  bacterium  tuberculosis,  show 
great  resistance  to  autolysis,  and  in  consequence  culture 
filtrates  contain  only  a  small  quantity  of  poisonous 
substances. 

The  poisonous  substances  found  in  culture  filtrates 
were  at  first  assumed  to  be  secretory  products  of  the 
bacteria,  and  were  regarded  as  true  toxin  formations. 
If  we  characterize  toxins  as  having  the  property,  in  def- 
inite doses,  of  bringing  about  the  death  of  an  animal,  we 
find  that  the  endotoxins  also  have  this  property.  We 
have  also  characterized  the  toxins  with  the  property  of 
bringing  about  an  active  immunity  in  animals  when  they 
are  injected  in  sublethal .  closes,  and  that  the  serum  of 
such  animals  when  injected  into  normal  animals  protects 
them  against  the  minimum  fatal  dose  of  the  toxin — that 
is,  it  brings  about  a  passive  immunity.  On  the  other 
hand,  the  injection  of  the  endotoxins  results  in  the  induc- 
tion of  bactericidal  immunity  with  the  formation  of 
amboceptors  of  a  specific  character. 

THE  POIXT  OF  ACTION  OF  TOXIN  AND  ENDO- 
TOXIN. — With  regard  to  the  point  of  action  of  the 
toxins  the  experiments  of  Wasserman  and  others  in- 
dicate that  as  far  as  tetanus  toxin  is  concerned  this 
poison  has  a  direct  affinity  for  nerve  cells.  With  regard 
to  diphtheria  toxin  one  sees,  on  making  a  postmortem 


570  BA  CTERIOLOG  Y. 

examination  of  an  animal  injected  with  the  toxin,  certain 
characteristic  lesions  which  have  been  brought  about  by 
the  action  of  the  toxin.  For  instance,  there  is  always 
very  marked  hypersemia  and  necrosis  at  the  point  of 
injection.  Certain  internal  organs  also  show  almost 
constantly  marked  hypersemia,  especially  the  adrenals 
and  the  chain  of  hsemolymph  glands  lying  in  the  retro- 
peritoneal  space.  Histological  studies  of  the  different 
tissues  have  revealed  certain  other  lesions  attributable 
to  the  action  of  the  toxin,  especially  areas  of  necrosis  in 
the  liver  and  other  organs.  Our  knowledge  of  the  man- 
ner in  which  sudden  death  is  frequently  brought  about 
during  convalescence  from  diphtheria  shows  that  the 
fatal  result  in  such  conditions  is,  however,  not  due  to 
any  of  the  alterations  or  reactions  of  the  toxin  that  have 
so  far  been  considered.  Death  during  the  period  of  con- 
valescence from  diphtheria  is  due  to  degenerations  occur- 
ring in  the  nerve  trunks  of  important  nerves,  especially 
of  the  pneumogastric.  With  this  fact  in  mind  it  seems 
evident  that  we  must  regard  this  form  of  toxic  action  of 
diphtheria  toxin  as  of  far  greater  moment.  In  poison- 
ing with  diphtheria  toxin  then  we  also  see  secondarily 
at  least,  if  not  primarily,  a  direct  affinity  of  the  toxin 
for  nerve  cells. 

With  regard  to  the  action  of  the  endotoxins  the  facts 
at  hand  are  far  less  satisfactory.  According  to  Ehrlich's 
side-chain  theory  the  amboceptors  formed  in  the  serum 
as  the  result  of  recovery  from  infection  have  been  formed 
as  the  result  of  the  overproduction  of  certain  cell  recep- 
tors, because  of  the  action  of  the  bacteria  themselves  or 
their  endotoxins  upon  certain  tissue  cells.  When  we 
come  to  look  for  evidence  as  to  the  particular  organs  or 
cells  that  are  aifected  in  different  infections  we  find  very 


INFECTION  AND  IMMUNITY.  571 

little  that  is  conclusive,  except  the  fact  that  Metchnikoff 
has  repeatedly  claimed  that  in  all  of  these  infections  the 
leucocytes  are  alone  the  cause  of  the  complicated  appear- 
ances resulting  from  infection.  Wolff1  is  inclined  to 
believe  that  the  antibody  formation  occurs  in  all  other 
organs  except  the  one  in  which  the  endotoxin  exerts  its 
poisonous  action,  because  Pfeiffer  and  Marx,  and  Wasser- 
man  in  their  work  upon  microspira  comma  and  bacillus 
typhosus  have  shown  that  the  principal  points  of  forma- 
tion of  the  specific  immune  body  are  the  haematopoietic 
organs.  Wolff  is  inclined  to  regard  the  principal  point 
of  attack  of  the  endotoxins  of  all  pathogenic  organisms 
to  be  the  central  nervous  system,  and  as  the  result  of 
certain  experiments  which  he  has  made  he  believes  that 
the  immune  bodies  are  formed  in  the  other  organs.  The 
facts  which  have  led  him  to  this  conclusion  are  the  re- 
sults obtained  in  intracerebral  injections  of  certain  poisons. 
Such  injections,  of  course,  bring  the  poison  in  direct  re- 
lation with  the  point  of  selective  action,  and  there  is 
little  opportunity  for  the  anchoring  or  destruction  of  the 
poison  by  other  tissue  cells  or  body  fluids.  In  injections 
into  other  parts  of  the  body,  especially  when  endotoxins 
or  bacteria  are  employed,  there  is  an  opportunity  for  the 
anchoring  of  these  elements  to  the  receptors  of  certain 
cells  and,  in  consequence,  unless  the  amount  of  toxin  is 
very  large,  the  formation  of  antibodies  takes  place.  On 
the  basis  of  such  experimental  results  it  seems  probable 
that  Wolff's  conclusions  are  correct. 

THE  DEFENCE  OF  THE  BODY  AGAINST  INFEC- 
TION.— Briefly  stated,  the  invasion  of  the  body  by  infec- 
tive micro-organisms  may  best  be  conceived  as  a  contest 

1  Wolff:  Centralblatt  f.  Bacteriologie,  originate,  1904,  Bd.  37,  and 
Berliner  klin.  Wochenschr.,  1904. 


572  BACTERIOLOGY. 

between  the  invading  organisms  on  the  one  side  and 
the  resisting  tissues  of  the  animal  body  on  the  other ; 
the  weapons  of  offence  of  the  former  being  the  poison- 
ous products  of  their  growth,  while  the  means  of 
defence  possessed  by  the  latter  are  the  phagocytic  cells, 
such  as  the  leucocytes,  the  large  mononuclear  cells  of 
the  blood,  and  the  connective  tissue  and  endothelial  cells, 
as  well  as  the  vital  substances  which  act,  so  to  speak,  as 
antidotes  to  bacterial  poisons.  If  the  leucocytes  and 
tissue  elements  are  not  of  sufficient  vigor  to  destroy  the 
invading  bacteria  or  to  render  inert  the  poisons  pro- 
duced by  them,  the  bacteria  are  victorious  and  infection, 
in  different  degrees  of  severity,  results ;  on  the  other 
hand,  if  there  be  failure  to  excite  disease,  the  tissues  are 
victorious,  and  are  then  said  to  be  resistant  to  or  immune 
from  this  particular  type  of  infection. 

In  some  cases  the  protective  bodies  possessed  by  the 
animal  act  directly  upon  the  invading  organisms  them- 
selves— i.  e.j  they  are  germicidal ;  in  others  their  func- 
tion is  more  that  of  antidotes,  or  neutralizers  in  the 
chemical  sense,  of  the  poisons  produced  by  these  organ- 
isms, the  organisms  themselves,  in  certain  instances, 
experiencing  only  slight  injury  from  a  limited  sojourn 
in  the  living  tissues.  For  those  constituents  of  the 
animal  body  that  are  by  nature  endowed  with  germi- 
cidal peculiarities,  the  designations  "alexins"  (Buchner) 
and  "defensive  proteids"  (Hankin)  have  been  sug- 
gested. Careful  investigation  has  shown  that  the  normal 
bactericidal  properties  of  the  blood  serum  rest  upon  the 
presence  of  immune  substances  similar  in  nature  to  those 
found  in  the  blood  serum  of  immune  animals — i.  e.,  (I)  a 
specific  immune  body  (intermediary  body)  for  a  partic- 
ular organism,  and  (2)  complement.  So  far  as  we  can 


• 

INFECTION  AND  IMMUNITY.  '573 

learn  the  blood  serum  contains  normally  a  small  amount 
of  antitoxic,  agglutinative,  and  bactericidal  action  against 
a  great  variety  of  pathogenic  bacteria. 

To  those  ill-defined  substances  whose  affinities  are 
restricted  to  the  soluble  toxins  elaborated  by  the  inva- 
ding bacteria  the  name  "  antitoxins "  is  now  generally 
applied.  Contrary  to  what  we  have  seen  in  the  case  of 
the  "  alexins  "  normally  present,  antitoxins  are  to  be  de- 
tected in  the  normal  animal  organism  in  very  small 
amounts.  When  they  do  exist  under  such  conditions 
they  are  of  but  comparatively  feeble  potency.1  In  the 
great  majority  of  instances  antitoxic  activities  are  acquired 
peculiarities ;  acquired  in  some  cases  in  a  more  or  less  nat- 
ural manner,  as  in  the  course  of  a  non-fatal  attack  of  a 
specific  malady ;  induced  in  others  by  purely  artificial 
means,  as  we  have  seen  to  be  possible  in  the  case  of  diph- 
theria, tetanus,  etc.  Our  acquaintance  with  these  bodies 
extends  little  further  than  their  physiological  functions 
and  some  of  the  means  that  induce  their  generation. 
We  have  no  satisfactory  knowledge  of  their  intimate 
nature  or  of  the  primary  sources  of  their  production. 
They  are  believed  by  some  (Buchner 2  and  Metchnikoif3) 
to  represent,  when  artificially  induced,  bacterial  toxins 
that  have  been  modified  by  the  vital  action  of  the  inte- 
gral cells  of  the  body  ;  and  Koux 4  and  Buchner 5  main- 

1  See  Bolton  :  Transactions  of  Association  of  American  Physicians, 
1896,  vol.  xi.  p.  62.  Pfeiffer :  Deutsche  med.  Wochenschrift,  1896, 
No.  8.  Fischl  and  v.  Wunschheim:  Centralblatt  fur  Bakteriologie, 
Pnrasitenkunde,  und  Infektionskrankheiten,  1896,  A bt.  i.  Bd.  xix.  S. 
<>."">:>.  Wassermann  :  Berliner  klin.  Wochenschrift,  1898,  No.  1. 

'Bur-liner:  Miinchener  raed.  Wochenschrift,  1893,  Nos.  24  and  25. 

3Metschnikoff:  Weil's  Handbuch  der  Hygiene,  Bd.  ix.  Lieferung  1, 
S.  48. 

*Roux  :  Annales  de  1'Institut  Pasteur,  1894.  p.  722. 

5 Buchner:  Berliner  klin.  Wochenschrift,  1894,  No.  4. 


574  BACTERIOLOGY. 

tain  that  they  exhibit  their  protective  functions  less  by 
direct  combination  with  the  toxins  than  by  a  specific 
stimulation  of  the  tissue-cells  that  enables  the  latter  to 
resist  the  harmful  influences  of  the  poisonous  bacterial 
products.  On  the  other  hand,  Behring,1  Ehrlich,2  and 
their  associates  contend  that  they  are  vital  tissue  ele- 
ments, having  the  property  of  combining  directly  with 
the  toxins  to  form  "  physiologically  neutral "  toxin-anti- 
toxin compounds  that  are  in  a  manner  analogous  to  the 
double  salts  of  certain  chemical  reactions. 

NATURAL,  IMMUNITY. — It  is  well  known  that  among 
man  and  the  lower  animals  individuals  are  frequently 
encountered  who  are,  in  general,  less  susceptible  to  infec- 
tion than  are  others  of  their  species ;  and  that  particu- 
lar species  of  animals  not  only  do  not  suffer  naturally 
from  certain  specific  diseases,  but  resist  all  efforts  to 
produce  the  diseases  in  them  by  artificial  methods ;  in 
other  words,  they  are  naturally  immune  from  them. 
The  term  "  natural  immunity,"  as  now  employed, 
implies  a  congenital  condition  of  the  individual  or 
species,  a  condition  peculiar  to  his  idioplasm,  which  has 
been  transmitted  to  him  as  a  tissue-characteristic  through 
generations  of  progenitors. 

ACQUIRED  IMMUNITY. — Again,  it  is  often  observed 
that  an  individual  or  an  animal  after  having  recovered 
from  certain  forms  of  infection  has  thereby  acquired 
protection  from  subsequent  attacks  of  like  character;  in 
other  words,  they  are  said  to  have  acquired  immunity 
from  this  disease.  "Acquired  immunity"  implies, 
therefore,  a  condition  of  the  tissues  of  an  individual, 

1  Behring :  Infektion  und  Desinfektion,  Leipzig,  1894,  S.  248. 

2  Ehrlich :  Klinisches  Jahrbuch,  1897,  Bd.  vi.  Heft  2,  S.  311.     Fort- 
schritte  der  Medicin,  1897,  Bd.  xv.  No.  2, 


INFECTION  AND  IMMUNITY.  575 

not  of  necessity  peculiar  to  other  members  of  the  race 
or  species,  that  has  originated  during  his  life  from  the 
stimulation  of  his  integral  cells  by  one  or  another  of  the 
specific  infective  irritants  that  may  have  been  purposely 
introduced,  or  accidentally  gained  access  to  his  body. 

Active  Immunity. — Acquired  immunity  may  be  either 
active  or  passive  in  character.  Active  immunity  is 
that  form  which  results  from  recovery  from  infection 
acquired  in  a  natural  way,  or  from  infection  induced  by 
the  injection  of  dead  or  living  organisms  as  a  prophy- 
lactic measure  against  infection. 

Passive  Immunity. — Passive  immunity  is  that  form 
in  which  the  immune  bodies  generated  in  a  susceptible 
animal,  as  the  result  of  systematic  injections  of  dead  or 
living  cultures,  are  introduced  into  a  human  being 
to  protect  against  infection.  The  antitoxic  serums  have 
been  employed  most  frequently  to  bring  about  passive 
immunity.  The  protective  value  of  diphtheria  anti- 
toxin in  those  that  have  been  exposed  to  infection  is 
well  established.  The  use  of  tetanus  antitoxin  for  pro- 
phylactic purposes  is  also  recommended  in  cases  where 
there  is  a  possibility  of  the  development  of  tetanus. 

VACCINATION  AGAINST  BACTERIAL  DISEASES. — 
The  employment  of  various  prophylactic  measures 
against  infectious  diseases  has  received  much  attention 
in  recent  years.  The  measures  employed  in  different 
diseases  vary  somewhat,  though  in  general  the  principles 
are  similar. 

The  first  measures  of  this  nature  that  were  employed 
on  a  large  scale  are  those  of  the  Haffkine  vaccination 
against  cholera  and  plague  by  means  of  cultures  that 
had  been  killed  after  heating  to  a  moderate  temperature. 
The  dead  organisms  when  injected  bring  about  a  reac- 


576  BACTERIOLOGY. 

tion  in  the  body  as  shown  by  a  marked  increase  in  the 
agglutinative  and  bactericidal  properties  of  the  blood 
serum  against  the  particular  organism.  The  favorable 
results  following  the  use  of  the  Haff  kine  fluid  in  pre- 
venting plague  have  already  been  given  (see  p.  325). 

AYright  has  introduced  a  similar  method  of  vaccina- 
tion against  typhoid  fever.  The  prophylactic  treatment 
consists  of  one  or  two  injections  of  dead  cultures  of 
bacillus  typhosus.  Caiger l  gives  the  results  obtained 
in  the  British  regiments  serving  in  India.  Amongst 
15,384  inoculated  men  the  incidence  of  typhoid  fever 
was  0.8  percent.,  as  against  1.5  per  cent,  in  the  uninocti- 
lated.  The  case  mortality  amongst  the  inoculated  was 
15.6  per  cent.,  as  against  26.6  per  cent,  in  the  uninocu- 
lated.  The  results  obtained  in  the  military  hospitals  in 
South  Africa  show  that  the  case  mortality  was  8.2  per 
cent,  among  the  inoculated,  as  against  15.1  per  cent, 
among  the  uninoculated ;  a  reduction  in  the  mortality 
of  about  50  per  cent.  In  the  staffs  of  three  of  the  mili- 
tary hospitals  the  reduction  in  the  mortality  was  nearly 
threefold. 

Bresredka 2  sought  to  find  a  method  of  immunization 
against  plague,  cholera,  and  typhoid  that  would  be  free 
from  the  objectionable  features  of  the  method  now  in 
use.  For  this  purpose  he  places  a  culture  of  bacil- 
lus typhosus  into  typhoid  immune  serum  until  it  is 
completely  agglutinated.  The  bacteria  are  then  removed 
from  the  serum  by  centrifuging  and  subsequently  washed 
with  sterile  physiological  salt  solution  in  order  to 
remove  all  trace  of  the  serum.  The  bacteria  are  then 
heated  to  60°  C.  for  several  hours.  When  these  bac- 

1  Caiger :  The  Lancet,  1904,  vol.  ii.,  p.  1467. 

2  Bresredka  :  Annals  of  the  Pasteur  Institute,  1902,  T.  16. 


INFECTION  AND  IMMUNITY.  577 

teria  are  now  injected  they  bring  about  a  distinct  immu- 
nity within  twenty-four  hours  without  inducing  any  of 
the  unfavorable  effects  seen  in  the  Haffkine  vaccination, 
such  as  fever,  pain,  weakness,  etc.  The  immunity 
induced  in  this  manner  rests  upon  the  formation  of 
specific  antibodies.  The  agglutinated  bacteria  can  be 
preserved  for  a  long  time.  The  immunization  is  not 
dangerous,  as  no  complications  have  been  noted. 

PRECIPITINS. — The  immunization  of  animals  with  a 
variety  of  substances  other  than  bacteria  has  served  to 
shed  light  upon  the  intimate  mechanism  of  immunity. 
One  of  the  reactions  that  is  noticed  as  the  result  of  such 
immunization  is  the  formation  of  precipitous  when  the 
serum  of  the  immunized  animal  is  mixed  with  the  sub- 
stance with  which  it  has  been  treated.  For  instance, 
the  serum  of  an  animal  that  has  received  repeated  injec- 
tions of  blood  from  an  alien  species,  will  cause  a  pre- 
cipitate to  form  when  mixed  with  the  serum  of  the 
species  of  animal  from  which  the  blood  has  been 
derived.  These  "  precipitins,"  as  they  are  called,  are 
specific  in  that  they  form  precipitates  only  with  the 
serum  of  the  species  of  animal  from  which  the  blood  has 
been  derived.  If  precipitates  are  formed  with  the  serum 
of  other  species  of  animals  it  is  always  in  very  much 
lower  dilutions. 

This  precipitin  reaction  is  so  characteristic  that  it  is 
now  employed  as  the  most  satisfactory  test  for  blood 
of  a  particular  species,  especially  in  medicolegal  cases 
requiring  the  differentiation  between  human  blood  and 
that  of  the  domestic  animals.  Albuminous  urine  or 
other  albuminous  fluids  may  also  be  employed  in  the 
immunization  of  the  animals  arid  similar  agglutinins  are 
formed  which  produce  precipitates  in  the  blood  serum, 
37 


578  BACTERIOLOGY. 

In  like  manner,  the  repeated  injection  of  milk  of  one 
species  of  animal  into  another  will  result  in  the  forma- 
tion of  precipitins  in  the  blood  serum  of  the  treated 
animal  that  will  precipitate  the  milk  of  that  species  of 
animal  from  which  the  milk  was  derived. 

AGGLUTININS. — In  acquired  immunity  as  the  result 
of  recovery  from  a  bacterial  disease,  and  in  induced 
immunity  after  repeated  injections  of  dead  or  living 
cultures  of  bacteria,  the  blood  serum  acquires  the  prop- 
erty of  agglutinating  the  bacteria  causing  the  infection. 
This  agglutination,  as  it  is  called,  is  brought  about  by 
the  presence  in  the  blood  serum  of  an  antibody  that  has 
the  property  of  bringing  about  the  clumping  of  the 
bacteria  and  causes  the  cessation  of  motility  in  suspen- 
sions of  motile  bacteria.  This  form  of  antibody  is 
spoken  of  as  "  agglutinin."  The  specific  action  of  the 
agglutinin  is  of  such  a  nature  that  it  has  for  a  long  time 
been  employed  as  a  means  of  diagnosing  certain  dis- 
eases, especially  typhoid  fever,  the  reaction  being  known 
under  the  name  of  Widal  reaction. 

It  has  been  found  that  normal  blood  serum  of  both 
man  and  the  domestic  animals  contains  normally  agglu- 
tinins  for  a  variety  of  bacteria  (Bergey).1  The  normal 
agglutinins  are  usually  present  in  relatively  low  amounts, 
though  occasionally  individuals  are  encountered  that 
possess  this  property  to  an  unusual  degree.  These 
"common"  agglutinins,  as  they  are  called,  may  be 
removed  from  the  serum  by  saturation  with  related 
organisms,  but  the  specific  agglutinins  resulting  from 
immunization  with  a  particular  organism  are  not  removed 
in  this  manner. 

The  agglutination  reaction  is  frequently  employed  for 

•     J  Bergey  :  Journal  of  Medical  Eesearch,  1903,  vol.  v.,  p.  21. 


INFECTION  AND  IMMUNITY.  579 

the  purpose  of  ascertaining  the  identity  of  bacteria,  and 
in  applying  this  diagnostic  test  for  this  purpose  it  is 
necessary  to  know  the  limits  of  the  agglutinating  power 
of  the  serum  for  the  organism  employed  in  immunizing 
the  animal,  so  as  not  to  be  misled  by  the  presence  of 
relatively  large  amounts  of  the  common  agglutinins.1 

The  agglutinating  properties  of  the  blood  serum  of 
an  immune  animal  are  not  always  in  proportion  to  its 
protective  or  curative  properties.  There  may  be  a  rela- 
tively high  degree  of  agglutination  without  a  corre- 
sponding bactericidal  action  of  the  serum.  From  this 
fact  the  agglutinating  properties  of  a  serum  cannot  be 
taken  as  a  basis  of  its  value  when  employed  therapeu- 
tically.  The  exact  relation  between  the  agglutinating 
and  the  bactericidal  properties  of  the  blood  serum  of  an 
animal  cannot  be  stated,  though  it  seems  probable  that 
they  are  both  the  result  of  certain  reactive  processes  on 
the  part  of  the  tissue-elements  against  the  bacteria. 

THE  MECHANISM  OF  IMMUNITY. — The  problem 
involving  the  explanation  of  the  interesting  ideas  and 
observations  with  regard  to  immunity  has  afforded 
material  for  reflection  and  hypothesis  for  a  long  time. 
It  is  only  through  investigations  conducted  during  recent 
years  that  it  has  met  with  anything  approaching  satis- 
factory solution,  and  even  now  there  remain  a  number 
of  important  points  that  are  veiled  in  obscurity. 

Conspicuous  among  the  observers  who  have  endeav- 
ored to  explain  the  mechanism  of  immunity  may  be 
mentioned  Chauveau,  Pasteur,  Metchnikoff,  Buchner, 
Fliigge  and  his  pupils  (Smirnow,  Sirotinin,  Bitter, 

1  A  great  deal  of  work  has  been  done  in  recent  years  on  the  common 
and  specific  agglutinins  in  dysentery  immune  serum  by  Park  and  his 
associates,  as  well  as  by  others  (see  Journal  of  Medical  Research,  vols. 
v.,  vi.,  and  vii.). 


580  BA  CTERIOLOG  Y. 

Nuttall),  Fodor,  Hankin,  Pfeiffer,  Ehrlich,  Behring, 
Roux,  and  a  host  of  others  whose  names  are  more  or 
less  prominent  in  current  literature.  In  the  following 
pages  we  will  present  and  discuss  certain  results  of 
investigations  that  serve  both  to  mark  the  evolutionary 
progress  of  our  knowledge  and  to  elucidate  the  more 
important  features  of  this  complicated  subject, 

THE  RETENTION  HYPOTHESIS  OF  CHAUVEAU. — In 
1880  Chauveau1  suggested  an  explanation  for  the 
phenomenon  of  immunity  that  has  since  been  known  as 
the  " retention  hypothesis"  It  is,  in  short,  as  follows  : 
that  the  immunity  commonly  seen  to  exist  in  animals 
that  have  passed  through  an  attack  of  infection  from 
a  subsequent  outbreak  of  the  same  malady,  and  likewise 
the  immunity  that  has  been  produced  artificially  by 
vaccination,  exist  by  virtue  of  some  bacterial  product 
that  has  been  retained  or  deposited  in  the  tissues  of  those 
animals,  and  that  this  product  by  its  presence  prevents 
the  development  of  the  same  organisms  if  they  should 
subsequently  gain  access  to  the  body. 

Bearing  upon  this  view  the  experiments  of  Sirotinin,2 
made  with  cultures  of  various  pathogenic  bacteria, 
demonstrated  that,  in  so  far  as  culture-experiments  were 
concerned,  the  only  substance  produced  by  growing 
bacteria  that  could  be  in  any  way  inimical  to  their  further 
development  were  substances  that  gave  rise  to  altera- 
tions in  the  reaction  of  the  medium  in  which  they  were 
developing — i.  e.,  acids  or  alkalies  produced  by  the  bac- 
teria themselves.  So  long  as  the  organisms  were  not 
actually  dead  from  exposure  to  these  substances  correc- ' 
tion  of  the  abnormal  reaction  was  followed  by  further 
development  of  the  organisms.  Sirotinin  also  states 

1  Comptes  rendus,  etc.,  July,  1880,  No.  91. 

2  Zeitschrift  fur  Hygiene,  1888,  Bd.  iv. 


INFECTION  AND  IMMUNITY.  581 

that  materials  containing  the  products  of  growth  of 
bacteria,  so  long  as  they  are  maintained  at  a  neutral  or 
only  slightly  alkaline  reaction,  serve  very  well  as  media 
upon  which  to  cultivate  again  the  same  organism  that 
produced  them,  providing  the  nutritive  elements  have 
not  been  entirely  exhausted.  He  remarks  that,  if  in  such 
a  concentrated  form  as  we  find  the  life-products  of  bac- 
teria in  the  medium  in  which  they  are  growing,  no  inhib- 
itory compounds  other  than  acids  and  alkalies  are  to 
be  detected,  it  is  hardly  probable  that  they  are  produced 
in  the  tissues  of  the  living  animal,  and  retained  there 
intact,  to  a  degree  sufficient  to  prevent  the  growth  of 
bacteria  that  may  subsequently  gain  entrance  to  these 
tissues,  after  the  disappearance  of  the  organisms  concerned 
in  the  primary  invasion.  Oh  the  other  hand,  Salmon 
and  Smith,1  Roux  and  Chamberland,2  and  others  had 
demonstrated  that  a  sort  of  immunity  against  certain 
forms  of  infection  may  be  afforded  to  susceptible  ani- 
mals by  the  injection  into  their  tissues  of  the  products 
of  growth  of  particular  organisms  which  would,  if  them- 
selves introduced  into  the  animal  body,  produce  fatal 
results.  Though  this  observation  of  Salmon  and  Smith 
attracted  comparatively  little  attention  at  the  time  it 
was  made,  it  serves,  nevertheless,  as  we  shall  see  sub- 
sequently, as  the  starting-point  for  a  line  of  investigation 
that  has  furnished  practically  all  the  information  of  im- 
portance that  we  possess  on  this  complicated  subject. 

THE  EXHAUSTION  HYPOTHESIS  OF  PASTEUR. — 
As  opposed  to  the  view  of  Chauveau,  Pasteur3  and 
certain  of  his  pupils  believed  that  the  immunity  fre- 

1  Proceedings  of  the  Biological  Society,  Washington,  D.  C.,   1886, 
vol.  iii. 

2  Anuales  de  Tlnstitut  Pasteur,  1888-'89,  tomes  i.,  ii. 

3  Bulletin  de  1' Academic  de  Medecine,  1880. 


582  BACTERIOLOGY. 

quently  afforded  to  the  tissues  by  an  attack  of  infection 
or  following  upon  vaccination  against  infection,  was 
due  rather  to  an  abstraction  from  the  tissues,  by  the 
organisms  that  were  concerned  in  the  primary  attack, 
of  a  something  that  is  necessary  to  the  growth  of  the 
infecting  organism  should  it  gain  entrance  to  the  body 
at  any  subsequent  time.  This  view  is  known  as  the 
"  exhaustion  hypothesis" 

As  to  the  exhaustion  hypothesis  of  Pasteur,  there  is, 
as  yet,  no  evidence  whatever  for  its  support ;  and,  in 
fact,  in  the  light  shed  by  more  recent  investigations  this 
is  probably  the  least  tenable  of  any  of  the  several  hy- 
potheses advanced  in  explanation  of  immunity.  The 
work  of  Bitter,1  which  was  undertaken  with  the  view 
of  determining  if,  in  the  process  of  acquiring  immunity, 
there  occurred  this  exhaustion  from  the  tissues  of  mate- 
rial necessary  to  the  growth  of  bacteria  that  might  gain 
entrance  to  them  at  some  later  date,  gave  only  negative 
results.  The  flesh  of  animals  in  which  immunity  had 
been  produced  contained  all  the  elements  necessary  for 
the  growth  and  nutrition  of  the  bacteria  against  which 
the  animals  had  been  protected,  just  as  did  the  flesh  of 
non-vaccinated  animals. 

THE  PHAGOCYTOSIS  THEORY  OF  METCHNIKOFF. 
— In  1884  Metchnikoff 2  published  the  first  of  a  series 
of  observations  upon  the  behavior  of  certain  of  the 
mesodermal  cells  of  lower  animals  toward  insoluble 
particles  that  may  be  present  in  the  tissues  of  these 
animals.  The  outcome  of  these  investigations  was  the 
establishment  of  his  well-known  doctrine  of  phago- 
cytosis, the  principle  of  which  is  that  the  wandering 

1  Zeitschrift  fur  Hygiene,  1888,  Bd.  iv. 

2  Arbeiten  aus  dem   Zoologischen    Institut  der  TJniversitat  Wien, 
1884,  Bd.  v.     Fortschritte  der  Medicin,  1884,  Bd.  ii. 


INFECTION  AND  IMMUNITY.  583 

cells  of  the  animal  organism,  more  especially  the  leuco- 
cytes, possess  the  property  of  taking  up,  rendering 
inert,  and  digesting  micro-organisms  which  they  may 
encounter  in  the  tissues.  Metchnikoff  believed  that  in 
this  way  immunity  from  infection  may  in  many,  if  not 
all,  cases  be  explained.  He  believed  that  suscepti- 
bility to,  or  immunity  from,  infection  was  essentially  a 
matter  between  the  invading  bacteria  on  the  one  hand, 
and  the  leucocytes  and  the  tissue  cells  on  the  other. 
The  success  or  failure  of  the  leucocytes  in  protecting 
the  animal  against  infection  depends,  according  to  this 
doctrine,  entirely  upon  the  efficiency  of  the  provisions 
possessed  by  them  for  destroying  bacteria,  and  upon  the 
aggressive  powers  of  the  invading  organisms.  When 
the  activity  of  the  body  cells  is  of  sufficient  vigor  to 
bring  about  the  death  of  the  bacteria  the  tissues  are 
victorious  ;  but  when  the  poisons  generated  by  the  bac- 
teria are  potent  to  arrest  the  phagocy  tic  action  of  the  leu- 
cocytes then  the  tissues  succumb  and  infection  results. 

THE  ALEXIN  THEORY  OF  BUCHNER. — Attractive  as 
this  doctrine  is,  plausible  as  were  the  arguments  in  sup- 
port of  it,  it  is  nevertheless,  in  the  light  of  later  evi- 
dence, inadvisable  to  accept  it  unconditionally  in  the 
sense  in  which  it  was  originally  propounded ;  in  fact, 
Metchnikoff  himself  has  in  recent  years  seen  fit  to  adopt 
certain  modifications  of  his  views  as  first  expressed. 
The  later  studies  of  a  number  of  investigators  indicate 
that  while  the  leucocytes  play  an  important  part  in  the 
phenomenon  of  immunity,  especially  in  natural  immu- 
nity, it  is  hardly  likely  that  this  always  occurs  through 
their  taking  up  and  digesting  within  themselves  inva- 
ding bacteria,  as  Metchnikoff  believed  ;  but  rather  that 
their  part  in  the  process  is  to  secrete  protective  chemical 


584  BACTERIOLOGY. 

substances  that  are  thrown  into  the  circulating  blood, 
and  which,  in  part  at  least,  comprise  the  defensive  bodies 
to  which  Buchner  has  given  the  name  "  alexins."  1 

The  first  severe  blow  that  Metchnikoff 's  theory  of 
phagocytosis  received  was  given  by  Nuttall,2  in  his 
work  upon  the  bactericidal  property  of  the  animal  econ- 
omy. In  these  experiments  Nuttall  demonstrated  that 
the  destruction  of  virulent  bacteria  in  the  blood  of 
animals  was  not  necessarily  dependent  upon  the  actual 
presence  of  living  leucocytes,  but  that  the  serum  of  the 
blood  when  quite  free  from  cellular  elements  possessed 
this  power  to  a  degree  equal  to  that  of  the  blood 
when  all  the  constituent  parts  were  present.  In  the 
blood,  as  such,  phagocytosis  could  be  seen  ;  but,  as  a 
rule,  the  bacteria  observed  within  leucocytes  presented 
evidence  of  having  undergone  degenerative  changes  be- 
fore they  had  been  taken  up  by  the  wandering  cells — /.  e, 
the  bacteria  had  evidently  been  injured  or  killed  by  the 
fluids  before  they  were  attacked  by  the  phagocytes. 

There  is  a  bit  of  interesting  history  leading  up  to  the 
idea  on  which  Nuttall  worked.  Contrary  to  the  beliefs  in 
existence  at  the  time,  Traube  and  Gscheidlen,3  as  far  back 
as  1874,  demonstrated  that  considerable  quantities  of  sep- 
tic material  could  be  injected  into  the  circulation  of  warm- 
blooded animals  without  apparently  any  effect  upon  the 
animal.  Particularly  was  this  the  case  with  dogs.  If 
they  injected  into  the  circulation  of  a  dog  as  much  as 
1.5  c.c.  of  decomposing  fluid,  blood  drawn  from  the  ani- 
mal after  from  twenty-four  to  forty-eight  hours  showed 
no  special  tendency  to  decompose,  though  it  was  kept 

1  See  Halm :  Archiv  fur  Hygiene,  1895,  Bd.  xxv.  S,  105. 

2  Zeitschrift  fur  Hygiene,  1888,  Bd.  iv. 

3  Jahresb.  der  Schlesischen  Ges.  fur  Cultur.,  1874 ;  Jahr.  iii.  p.  179. 


INFECTION  AND  IMMUNITY.  585 

under  observation  for  a  long  time.  As  a  result  of  these 
experiments,  the  question  that  naturally  presented  itself 
was :  Does  the  animal  organism  possess  the  power  of 
rendering  septic  organisms  inert,  and  if  so,  to  what 
extent?  They  believed  this  power  of  rendering  living 
organisms  inert  to  be  possessed  by  the  circulating 
blood  to  only  a  limited  degree,  for  after  the  injection 
of  much  larger  amounts  of  the  putrid  fluid  into  the 
blood  of  the  animal  death  usually  ensued  in  from 
twenty-four  to  forty-eight  hours.  The  blood  drawn 
from  the  animal  just  before  death  contained  the  living 
bacteria  of  putrefaction  and  promptly  underwent  decom- 
position. They  attributed  the  germicidal  phenomenon 
to  the  action  of  the  "  ozonized  oxygen  of  the  corpuscles 
of  the  blood." 

Similar  observations  were  made  in  1885  by  Fodor,1 
who  remarks  upon  the  rapidity  with  which  living  bac- 
teria disappear  from  the  circulating  blood  of  animals ; 
and  AVyssokowitsch,2  who  endeavored  to  explain  this 
disappearance 'experimentally,  went  wide  of  the  mark 
by  concluding  that  they  were  filtered  from  the  blood 
and  digested  by  the  parenchymatous  tissues. 

In  1882  Rauschenbach 3  demonstrated  that  when  in 
the  process  of  coagulation  fibrin  was  formed,  it  was  not 
as  a  specific  product  of  the  action  of  the  colorless  ele- 
ments of  the  blood  alone,  but  also  as  a  result  of  the 
combined  action  between  all  animal  protoplasms  and 
healthy  blood-plasma,  and  that  in  the  process  there  was 
always  a  disintegration  of  the  leucocytes  present.  In 

1  Fodor :  Archiv  fur  Hygiene,  Bd.  iv.  S.  129. 

2  Wyssokowitsch :  Zeitschrift  fur  Hygiene,  1886,  S.  3. 

3  Uober  die  Wechselwirkung  zwischen  Protoplasma  und  Blutplasma. 

Dissertation,  Dorpat,  1882. 


586  BACTERIOLOGY. 

1884  Groth1  demonstrated  further  that  such  a  disinte- 
gration of  leucocytes  occurred  in  normal  circulating 
blood,  though  here  it  was  not  accompanied  by  coagula- 
tion. The  results  of  these  observations  suggested  the 
question  :  Does  such  a  disintegration  occur  when  vege- 
table protoplasm  is  introduced  into  the  blood  ? 

For  the  purpose  of  answering  this  question,  Grohmann,2 
a  pupil  of  Alexander  Schmidt,  undertook  to  study  the  ac- 
tion of  the  circulating  blood  upon  the  vegetable  proto- 
plasm of  bacteria.  He  noticed  that  clotting  of  the  blood 
of  the  horse  was  very  much  accelerated  by  the  addition  to 
it  of  certain  bacteria ;  that  at  the  same  time  the  develop- 
ment of  the  bacteria  was  checked,  and  in  the  case  of  the 
pathogenic  varieties  their  virulence  was  diminished. 
This  was  particularly  the  case  when  the  anthrax  bacil- 
lus was  employed. 

Grohmann  seems  to  have  appreciated  the  significance 
of  this  observation,  though  he  made  no  attempt  to  study 
the  subject  more  closely.  He  remarks  that  the  system 
probably  possesses,  in  the  plasma  of  the  blood,  a  body  hav- 
ing disinfectant  properties.3  His  observations  on  this 
particular  point  were,  however,  only  incidental,  and  it 
was  not  until  the  researches  of  Nuttall,  directed  especi- 
ally toward  the  question  of  germicidal  properties  of 
normal  animal  fluids,  that  a  complete  and  acceptable 
demonstration  of  this  important  function  was  forthcom- 
ing, and  that  a  general  interest,  commensurate  with  its 
importance,  was  created  in  the  subject. 

1  Ueber  die  Schicksale  der  farblosen  Elemente  in  kreisendem  Blut. 
Dissertation,  Dorpat,  1884. 

2  Ueber    die    Einwirkung   des  zellenfreien    Blutplasma  auf  einige 
pflanzliche  Mikro-organismen.     Dissertation,  Dorpat,  1884. 

3  Loc.  cit.,  pp.  6  and  33. 


INFECTION  AND  IMMUNITY.  587 

NuttalFs  results  received  confirmation  from  all  sides. 
Fodor,1  Buchner,2  Lubarsch,3  Nissen,4  Stern,5  Prudden,6 
Charrin  and  Roger/  and  many  others  continued  in  the 
same  line,  and  all  made  practically  the  same  observation. 

After  the  demonstration  by  Nuttall  that  the  serum 
of  the  blood  was  directly  detrimental  to  the  vitality  of 
certain  pathogenic  bacteria,  a  number  of  investigators 
undertook  to  determine  the  conditions  most  favorable  to 
the  exhibition  of  this  phenomenon,  and  further  to  decide 
upon  the  constituent  of  the  serum  to  which  this  property 
is  due,  or  if  it  is  a  function  of  the  serum  only  as  a  whole. 

In  the  course  of  Buchner's  experiments  it  was  de- 
monstrated that  the  serum  was  robbed  of  this  property 
by  exposure  to  a  temperature  of  55°  C.  for  half  an 
hour ;  that  its  efficacy  as  a  germicide  was  not  dimin- 
ished by  alternate  freezing  and  thawing ;  that  by  dialy- 
sis or  extreme  dilution  with  distilled  water  its  germicidal 
activity  was  diminished  or  completely  checked ;  but 
that  an  equal  dilution  could  be  made,  if  sodium  chlo- 
ride solution  (0.6-0.7  per  cent.)  was  substituted  for  the 
distilled  water,  without  a  reduction  of  its  bactericidal 
activity.  From  this  he  concluded  that  the  active  ele- 
ment in  this  phenomenon  is  a  living  albumin,  an  essen- 
tial constituent  of  which  is  sodium  chloride,  and  which, 
when  robbed  of  this  salt  either  by  dialysis  or  dilution, 
becomes  inert  in  its  behavior  toward  bacteria.  For  this 

1  Central,  fur  Bakteriologie  und  Parasiteiikunde,  1890,  Bd.  vii.  No.  24. 

2  Archiv  fur  Hygiene,  1890,  vol.  x.  parts  1  and  2. 

3  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  1889,  Bd.  vi. 
No.  18. 

4  Zeitscrift  fur  Hygiene,  1889,  Bd.  vi.  part  3. 

5  Zeitscrift  fur  klin.  Medicin,  1890,  Bd.  viii.  parts  1  and  2. 

6 New  York  Medical  Rec;rd,  1890,  vol.  xxxvii.  pp.  85  and  86. 
7  Societe  de  Biologic  de  Paris. 


588  BACTERIOLOGY. 

or  these  germicidal  constituents  of  the  blood  he  sug- 
gested the  name  "  alexins." 

Buchner  found,  moreover,  that  the  activity  of  the 
serum  alone  against  bacteria  was  greater  than  when  the 
cellular  elements  of  the  blood  were  present.  This  he 
explains  by  the  assumption  that  in  the  serum  alone  the 
germicidal  element  predominates,  whereas  in  the  blood, 
as  such,  outside  of  the  body,  it  is  still  present,  but  its 
influence  is  counteracted  by  the  nutrition  offered  to  the 
bacteria  by  the  disintegrated  cellular  elements  ;  so  that 
here  the  nutritive  feature  is  most  conspicuous  and  the 
destructive  activity  toward  bacteria  is  less  effectual. 

A  closer  study  of  the  nature  of  this  germicidal  ele- 
ment in  the  body  of  animals  was  made  by  Hankin  and 
Martin.1  The  former  isolated  from  the  spleen  and 
lymphatic  glands  a  body — a  globulin — which  in  solu- 
tion possesses  germicidal  properties. 

Similar  germicidal,  ferment-like  globulins  have  been 
isolated  from  the  blood  by  Ogata,2  and  in  their  studies 
upon  tetanus  Tizzoni  and  Cattani 3  found  a  body  that 
was  antagonistic  to  the  poison  produced  by  the  organism 
of  this  disease. 

In  1890  Fodor4  showed  that  the  fatal  results  of 
anthrax  infection  could  be  materially  retarded  by 
alkalinization  of  the  circulating  blood ;  and  he  further 
states  that  it  is  occasionally  possible  by  the  admin- 
istration of  alkalies  per  os  to  rescue  animals  already 
infected. 

1  British  Medical  Journal,  May  31,  1890. 

2  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  1891,  vol.  ix, 
S.  599. 

3  Ibid.,  p.  685. 

*  Fodor :  Centralblatt  fur  Bakteriologie,  1890,  Bd.  vii.  S.  753-766. 


INFECTION  AND  IMMUNITY.  589 

According  to  the  observations  of  Vaughan,1  the  most 
important  germicidal  or  protective  agents  possessed  by 
the  body  are  the  nucleins ;  and  Kossel  has  shown  that 
the  cholera  vibrio,  streptococcus,  staphylococcus,  and 
typhoid  bacillus  are  destroyed  by  0.5  per  cent,  solution 
of  nucleinic  acid. 

Hankin  believed  the  globulins  or  "  defensive  pro- 
teids"  that  he  had  discovered  and  the  albuminoid 
bodies  studied  by  Buchner  to  be  identical.  The  most 
interesting  and,  in  the  light  of  work  that  has  appeared 
since,  the  most  important,  of  Hankin's  observations 
were  not  those  upon  the  power  of  these  globulins  to 
destroy  the  vitality  of  living  organisms,  but  rather 
those  upon  the  relation  between  them  and  the  poison- 
ous metabolic  products  of  growth  of  the  organisms. 
For  example,  if  the  poisonous  products  of  virulent 
anthrax  bacilli  be  isolated  and  mixed  with  the  glob- 
ulin extracted  from  normal  tissues,  the  experiments  of 
Hankin  showed  a  directly  destructive  action  on  the  part 
of  the  bacterial  products.  He  found  that  the  amount  of 
poisonous  albumose  produced  by  the  attenuated  anthrax 
bacilli  employed  as  vaccines  was  much  less  than  that 
produced  by  organisms  possessing  full  virulence ;  and 
he  suggested  that  perhaps  the  protective  influence  of 
vaccinations  that  are  practised  by  introducing  into  the 
animal  the  organisms  that  have  been  attentuated  in  vir- 
ulence is  due  to  a  gradual  tolerance  acquired  by  the 
cells  of  the  tissues  to  the  action  of  the  poison  when 
produced  in  these  small  quantities ;  in  the  same  way 
that  a  tolerance  was  acquired  by  the  tissues  for  the 
venom  of  the  rattlesnake  in  the  experiments  of  Sewall2 

1  Vaughan :  Medical  News,  May  20,  1893. 

2  Journal  of  Physiology,  1887,  vol.  vii.  p.  203. 


590  BACTERIOLOG  Y. 

(and  more  recently  in  the  work  of  Fraser,1  Calmette, 
Weir  Mitchell,  and  others),  and  similar  to  that  follow- 
ing the  injection  into  the  tissues  of  small  quantities  of 
hemialbumose,  which  in  large  amounts  rapidly  proves 
fatal. 

Of  utmost  importance  to  these  investigations  upon 
the  blood  and  fluids  of  the  body  are  the  experiments 
of  Behring  and  Kitasato  2  upon  the  production  of  im- 
munity from  tetanus.  In  their  studies  upon  the  blood 
of  animals  subjected  to  these  experiments  it  was  found 
that  it  was  not  only  possible  to  render  animals  immune 
from  this  disease,  but  that  the  serum  of  the  blood  of 
these  immunized  animals  afforded  immunity  when  in- 
jected into  the  peritoneal  cavity  of  other  animals  that 
had  not  been  so  protected ;  and  moreover,  that  in  some 
cases  this  serum  possesses  curative  powers  over  the 
disease  after  it  has  been  in  progress  for  a  time.  They 
found,  further,  that  the  serum  of  animals  that  had  been 
rendered  immune  from  tetanus,  when  brought  in  con- 
tact with  the  poison  of  tetanus,  completely  destroyed  its 
poisonous  properties,  and  that  the  serum  from  animals 
or  from  human  beings  that  do  not  possess  immunity 
from  this  disease  has  no  such  power. 

1  One  of  the  important  results  of  Eraser's  studies  is  the  demonstra- 
tion that  the  bile  of  normal  serpents  and  of  certain  warm-blooded 
animals,  notably  bovines,  rabbits,  and  guinea-pigs,  is  antitoxic  for 
serpent  venom.  The  antitoxic  value  of  the  bile  in  this  connection 
seems  to  be  fairly  comparable  to  that  of  other  antitoxins  obtained 
from  the  blood  of  artificially  immunized  animals.  Its  action  is  not 
only  prophylactic,  but  also  curative,  Fraser  having  succeeded  in 
rescuing  animals  by  the  injection  of  bile-extract  thirty  minutes  after 
they  had  received  what  would  otherwise  have  been  a  fatal  dose  of 
cobra  poison.  (See  Centralblatt  fur  Bakteriologie,  1897,  Bd.  xxii.  S. 
420.) 

'2  Behring  und  Kitasato :  Deutsche  med.  Wochenschrift,  1890,  Bd. 
xvi.  S.  1113. 


INFECTION  AND  IMMUNITY.  591 

The  demonstration  by  Behring  and  Kitasato  of  the 
fact  that  the  serum  of  an  immunized  animal  can  not 
only  confer  immunity  upon  another  susceptible  animal, 
but  in  the  case  of  tetanus  (and  diphtheria,  as  subse- 
quently demonstrated  by  Behring  and  his  associates), 
cure  the  disease  after  it  is  already  in  progress,  is  one  of 
the  most  important  steps  that  has  been  made  in  this 
entire  field  of  study.  The  application  of  the  principle 
involved  in  this  observation  to  the  cure  of  diphtheria 
in  man  has  resulted  in  a  triumph  which  marks  an  epoch 
in  modern  scientific  medicine.  The  same  principle  has 
been  employed  for  obtaining  curative  agents  against 
other  forms  of  infection  and  intoxication,  notably,  of 
Asiatic  cholera,  typhoid  fever,  lobar  pneumonia,  strep- 
tococcus and  staphylococcus  infection,  rabies,  tubercu- 
losis, bubonic  plague,  and  serpent-venom  ;  but  unfor- 
tunately, as  yet,  with  only  indifferent  success;  cer- 
tainly in  no  case  to  the  same  favorable  degree  as  has 
been  seen  in  the  treatment  of  diphtheria  with  antitoxic 
serum. 

Another  hypothesis  in  explanation  of  the  immunity 
acquired  by  the  tissues  of  the  animal  organism  is  that 
advanced  by  Buchner,1  who  suggests  that  in  the  primary 
infection,  from  which  the  animal  has  recovered,  there 
has  been  produced  a  reactive  change  in  the  integral  cells 
of  the  body  that  enables  them  to  protect  themselves 
against  subsequent  inroads  of  the  same  organism. 
Though  somewhat  more  vague  at  first  glance  than  the 
other  theories  in  regard  to  this  phenomenon,  it  has, 
nevertheless,  much  to  recommend  it,  and  in  the  light 
of  subsequent  research  is  regarded  by  many  as  prob- 

1  Buchner  :  Eiue  neue  Theorie  uber  Erzielung  von  Immunitat  gegen 
Infektionskrankheiten.  Muenchen,  1883. 


592  BACTERIOLOGY. 

ably  the  correct  explanation  of  the  establishment  of  im- 
munity in  a  number  of,  if  not  in  all,  cases.  Experiments 
that  bear  directly  upon  this  idea  have  demonstrated 
that,  if  animals  be  subjected  to  injections  of  the  poison- 
ous products  of  growth  of  certain  virulent  bacteria, 
they  respond  to  this  treatment  by  more  or  less  pro- 
nounced constitutional  reactions,  and  that  during  this 
period,  and  for  a  short  time  following,  they  are  protected 
from  the  invasion  of  the  virulent  bacteria  themselves. 
This  observation  has,  moreover,  not  been  confined  to 
those  cases  in  which  injections  of  the  products  of  growth 
were  followed  by  inoculations  with  the  bacteria  by 
which  they  were  produced,  but,  what  is  still  more  in- 
teresting and  confirmatory  of  Buchner's  view,  it  is 
claimed  that  a  sort  of  protection  from  certain  specific 
infections  can  also  be  afforded  to  animals  by  the  injec- 
tion into  them  of  cultures  of  entirely  different  species  of 
bacteria,  or  their  products,  and  that  in  some  cases  these 
are  not  of  necessity  of  the  disease-producing  varieties. 
For  instance,  Emmerich  and  Mattei1  claim  to  have 
rendered  rabbits  insusceptible  to  anthrax  through  injec- 
tions into  them  of  cultures  of  the  streptococcus  of  ery- 
sipelas. 

This,  they  claimed,  is  not  due  to  any  antagonism  be- 
tween the  two  organisms  themselves,  for  in  culture 
experiments  they  grew  well  together,  without  any 
alteration  in  their  pathogenic  properties;  but  rather 
to  the  induction  of  a  tissue  activity  by  which  resistance 
to  the  inroads  of  the  virulent  bacilli  was  established. 
Emmerich  and  Mattei  interpret  this  "reactive  tissue- 
change  "  as  a  power  acquired  by  the  integral  cells  of  the 
body,  through  the  influence  of  a  stimulus,  of  generating 

1  Emmerich  und  Mattei :  Fortschritte  der  Medicin,  1887,  S.  653. 


INFECTION  AND  IMMUNITY.  593 

a  product  that  is  detrimental  to  the  pathogenic  activity 
of  the  anthrax  bacilli.  In  a  later  paper1  Emmerich,  in 
association  with  Low,  offers  still  another  explanation  for 
the  phenomenon.  They  believe  that  the  protection 
afforded  an  animal  from  a  specific  infective  organism 
by  the  injection  into  it  of  another  organism  or  the 
products  of  its  growth  is  due  to  the  direct  bacteriolytic 
action  of  the  enzymes  peculiar  to  the  latter  upon  the 
former  species.  In  their  opinion,  the  enzymes  of  the 
latter  organism  enter  into  a  more  or  less  stable  combina- 
tion with  the  living  protoplasm,  and  in  this  state  are 
actively  destructive  (digestive)  for  the  invading  patho- 
genic species. 

Pawlowsky,2  who  obtained  similar  results  from  the 
introduction  into  animals  of  cultures  of  bacillus  pro- 
digiosuSj  of  micrococcus  aureus,  and  of  micrococcus  lan- 
ceolatus,  believes  them  to  be  due  to  the  induction  of 
increased  energy  on  the  part  of  the  wandering  cells, 
preparing  them  thus  for  the  difficult  task  of  destroying 
the  more  virulent  organisms  with  which  the  animal  is 
subsequently  to  be  inoculated. 

Protection  afforded  in  this  way  apparently  contra- 
indicates  a  specific  relation  between  the  morbific  ele- 
ments of  particular  infections  and  the  protective  sub- 
stances that  are  present  in  the  body  of  the  animal  that 
has  been  rendered  insusceptible  to  them.  It  is  proba- 
ble, however,  that  this  is  only  apparent,  and  that  the 
observations  of  Emmerich  and  Mattei  and  of  Paw- 
lowsky can  be  interpreted  in  another  way:  in  the  blood 
of  animals  there  is  present  what  may  be  termed  a  nor- 

1  Emmerich  und  Low :  Zeitschrift  fur  Hygiene  und  Infektionskrank- 
heiten,  1899,  Bd.  xxxi.  S.  1-5. 

2  Pawlowsky:  Virchow's  Archiv,  vol,  cviii.  p.  494, 

38 


594  BACTERIOLOGY. 

mal  protective  substance  (Buchner's  alexins)  having  no 
specific  relations  to  any  particular  variety  of  infection,  but 
offering  some  protection,  more  or  less  complete,  to  the 
animal  against  all  bacterial  invasion.  By  the  methods 
employed  in  the  preceding  experiments  it  seems  likely, 
in  the  light  of  more  recent  work,  that  this  normal  anti- 
dote was  simply  temporarily  accentuated  through  the 
tissue-stimulation  resultant  upon  the  treatment  that  the 
animals  had  received,  for  it  is  not  possible  to  bring 
about  in  this  way  as  high  or  as  permanent  a  degree  of 
immunity  in  an  animal  from  a  particular  disease  as  that 
which  can  be  obtained  by  the  use  of  the  specific  micro- 
organism causing  the  disease,  or  the  products  of  its 
growth,  especially  the  latter. 

A  striking  illustration  of  this  protective  reaction  on 
the  part  of  the  animal  tissues  is  brought  out  in  the 
course  of  R.  Pfeiifer's !  experiments  on  Asiatic  cholera. 
He  found  that  it  is  possible  to  confer  immunity  upon 
animals  from  this  disease;  that  the  blood-serum  of 
such  animals  protects  susceptible  animals  into  which 
it  is  injected  against  what  would  otherwise  be  a 
fatal  dose  of  the  cholera  spirillum ;  that  the  perito- 
neal fluid  of  the  artificially  immunized  animal  has 
an  almost  instantaneous  disintegrating  (bacteriolytic), 
bactericidal  action  upon  living  cholera  spirilla  that 
are  injected  into  the  peritoneal  cavity ;  that  the 
serum  from  the  immune  animal,  when  kept  for  a 
time,  has  no  such  effect  upon  cholera  spirilla  in 
the  test-tube ;  but  when  virulent  cholera  spirilla  are 
injected  into  the  peritoneum  of  an  animal  that  is 

1  Pfeiffer:  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  Bd. 
xviii.  S.  1 ;  Bd.  xx.  S.  198. 


INFECTION  AND  IMMUNITY.  595 

not  immune,  and  this  is  at  once  followed  by  an  intra- 
peritoneal  injection  of  serum  from  an  immune  animal, 
almost  instantly  the  peculiar  disintegration  of  the  bac- 
teria, as  observed  in  the  peritoneum  of  the  immune  ani- 
mal, can  be  detected.  This  latter  observation  is  of 
the  utmost  importance  in  its  bearing  on  Buchner's 
hypothesis,  for  we  see  here  a  serum  from  an  immune 
animal  that  is  capable  of  conferring  immunity  ;  capable, 
on  injection  into  a  susceptible  animal,  of  endowing  its 
fluids  with  the  peculiar  disintegrating,  germicidal 
function  noted  in  the  peritoneum  of  the  immune  ani- 
mal from  which  the  serum  originated  ;  quickly  loses  its 
bacteriolytic  activity  outside  the  body,  but  the  influ- 
ence of  which  in  the  peritoneum  of  a  susceptible  ani- 
mal is  to  call  forth  at  once  this  interesting  phenomenon. 
Manifestly  the  germicidal  substance  in  this  case  is 
either  generated  by  the  tissues  as  a  result  of  the  specific 
irritation  by  a  something  contained  in  this  serum — i.  e., 
in  consequence  of  a  reaction  on  the  part  of  the  peritoneal 
tissues,  or  possibly  those  of  the  entire  animal — or  else  it 
is,  as  Ehrlich  conceives  it  to  be,  a  complex  whose  physi- 
ological activity  depends  upon  the  union  of  at  least  two 
essential  groups — the  one  present  in  the  serum  of  the  im- 
mune animal,  and  the  other  in  the  fluids  of  the  normal 
animal. 

In  more  recent  investigations  Pfeiifer,  in  association 
with  Marx,  has  found  that  the  bactericidal  substances  in 
choiera-immune  animals  are  much  more  abundant  in 
the  blood-building  organs — spleen,  lymphatic  glands, 
and  bone-marrow — than  in  either  the  blood  or  other 
tissues.1 

1  Pfeiffer  and  Marx :  Deutsche  med.  Wochenschrift,  1898,  No.  3. 


596  BACTERIOLOG  Y. 

The  experiments  of  G.  and  F.  Klemperer '  upon  acute 
fibrinous  pneumonia,  though  too  limited  in  extent  to 
be  accepted  as  conclusive,  presented  nevertheless  a 
number  of  most  significant  suggestions,  not  only  in 
connection  with  several  obscure  features  of  this  disease, 
but  in  their  broader  bearing  upon  acquired  tissue-resist- 
ance in  general. 

These  authors  found  but  little  difficulty  in  conferring 
immunity  upon  animals  that  are  otherwise  susceptible  to 
the  pathogenic  action  of  the  organisms  concerned  in  the 
production  of  this  disease,2  by  the  introduction  into  their 
tissues  of  the  products  of  growth  of  the  organisms  from 
which  the  latter  had  been  separated.  The  immunity 
thus  produced  is  seen  in  some  cases  to  last  as  long  as 
six  months ;  again  it  is  seen  to  disappear  suddenly  in 
a  way  not  to  be  explained.  It  was  seen  in  one  case 
to  be  hereditary,  probably  having  been  transmitted  to 
the  young,  during  the  nursing-period,  through  the  rnilk 
of  the  mother,  as  Ehrlich3  has  shown  to  occur  in 
animals  artificially  immunized  from  abrin,  ricin,  and 
robin. 

The  energy  of  the  substance  that  has  the  power  of 
affording  immunity  was  seen  to  be  very  much  increased 
by  subjecting  it  to  temperatures  somewhat  higher  than 
that  at  which  it  was  produced  by  the  bacteria.  The 
Klemperers  found  that  if  this  substance  was  heated  to 

1  G.    and  F.  Klemperer  :  Berliner  klin.  Wochenschrift,   1891,  Nos. 
34  and  35. 

2  Animals  do  not,  as  a  rule,  present  the  pneumonic  changes  seen  in 
human  beings.     The  introduction  of  micrococcus  lanceolatus  into  their 
tissues  results,  in  the  case  of  susceptible  animals,  in  the  production  of 
septicaemia. 

3  Ehrlich  :  Zeitschrift  fiir  Hygiene  und  Infektionskrankheiten,  1892, 
Bd.  xii.  S.  183. 


INFECTION  AND  IMMUNITY.  597 

a  temperature  of  from  41°  to  42°  C.  for  three  or  four 
days,  or  to  60°  C.  for  from  one  to  two  hours,  its  intra- 
venous injection  was  followed  by  complete  immunity 
in  from  three  to  four  days ;  whereas,  if  the  un warmed 
material  was  used,  immunity  did  not  appear  before 
fourteen  days,  and  then  only  after  the  employment  of 
relatively  large  amounts.  Moreover,  when  the  pre- 
viously heated  products  are  introduced  into  the  circu- 
lation of  the  animal  the  systemic  reaction  is  of  but 
short  duration ;  but  if  the  un  warmed  substance  is 
employed,  immunity  is  manifest  only  after  the  onset 
of  considerable  elevation  of  temperature,  which  lasts 
for  a  long  time. 

In  explanation  of  these  differences  they  suggested 
that,  in  the  latter  case,  the  high  fever  that  is  seen  to 
occur  in  the  animal  may  serve  to  replace  the  warming 
to  which  the  bacterial  products  had  not  previously  been 
subjected,  and  which  is  necessary  before  they  are  in  a 
position  to  bring  about  the  condition  of  immunity. 
They  claimed  that  the  bacterial  products  employed  to 
produce  immunity  in  this  case  are  not,  in  reality,  the 
immunity-affording  substance,  but  that  they  are  only 
the  agents  that  bring  about  in  the  tissues  of  the  animal 
alterations  that  result  in  the  production  of  another  body 
that  protects  the  animal.  In  support  of  this,  their 
argument  was  that  several  days  are  necessary  for  the 
production  of  immunity  by  the  introduction  into  the 
animal  of  the  bacterial  products ;  whereas,  if  the  blood- 
serum  of  this  animal,  which  is  now  protected,  be  intro- 
duced into  the  circulation  of  another  animal,  no  such 
delay  is  seen,  but  instead  the  animal  is  forthwith  pro- 
tected. In  the  former  case  the  actual  protecting  body 
had  first  to  be  manufactured  by  the  tissues ;  whereas 


598  BA  CTERIOLOG  Y. 

in  the  second  it  is  already  prepared,  and  is  introduced 
as  such  into  the  second  animal. 

These  authors  found  the  serum  of  artificially  immun- 
ized animals  to  be  not  only  capable  of  rendering  other 
animals  immune,  but  to  be  possessed  of  curative  powers 
when  the  disease  was  already  in  progress.  The  serum 
of  immunized  animals  when  injected  into  the  circula- 
tion of  animals  in  which  there  was  a  body-temperature 
of  from  40.4°  to  41°  C.  reduced  this  temperature  to 
normal  (37.5°  C.)  in  twelve  consecutive  experiments 
during  the  first  twenty-four  hours  following  its  employ- 
ment. 

In  their  opinion,  the  crisis  seen  in  pneumonia  in 
human  beings  indicates  the  moment  at  which  the  pois- 
onous products,  manufactured  by  the  bacteria  located 
in  the  lungs,  are  present  in  the  circulation  in  amounts 
sufficient  to  stimulate  the  tissues  to  the  reaction  that 
results  in  the  production  of  the  antidotal  substance  that 
has  the  power  of  rendering  the  poisons  inert. 

At  the  time  of  the  crisis  in  pneumonia  the  bacteria 
themselves  are  in  no  way  affected.  They  remain  in  the 
lungs,  and  can  be  detected,  in  full  vigor  and  virulence, 
in  the  sputum  of  patients  a  long  time  after  the  disease 
is  cured.  They  have  lost  none  of  their  power  of  pro- 
ducing poisonous  products,  and  still  possess  their  orig- 
inal pathogenic  relations  toward  susceptible  animals. 
It  is  only  after  the  crisis  that  their  poisons  are  neutral- 
ized by  this  antidotal  proteid  that  has  been  produced 
by  the  cells  of  the  tissues,  and  as  this  occurs  the  sys- 
temic manifestations  gradually  disappear.  The  Klem- 
perers  claim  to  have  isolated  from  cultures  of  micro- 
coeeus  lanceolatus  a  proteid  body  that  is  the  agent  con- 
cerned in  producing  the  tissue-reaction  which  results  in 


INFECTION  AND  IMMUNITY.  599 

the  formation  of  the  protecting  substance.  They  like- 
wise isolated  from  the  serum  of  immunized  animals 
a  proteid  that  possessed  the  same  powers  as  the  serum 
itself,  viz.,  of  affording  immunity  and  curing  the 
disease. 

Here,  again,  it  appears  that  the  processes  of  infection 
and  immunity  are  chemical  in  their  nature,  the  active 
poisons  of  the  invading  organisms — "  the  pneumo- 
toxins  " — being  instrumental  in  producing  the  diseased 
condition,  while  the  antidotal  or  resisting  body  of  the 
tissues  —  "the  anti-pneumotoxin "  —  is  the  agent  by 
which  the  poison  is  neutralized. 

Results  in  general  analogous  to  those  of  G.  and  F. 
Klemperer  have  also  been  obtained  by  Emmerich  and 
Fowitzky.1 

In  the  light  of  these  experiments  the  hypothesis 
advanced  by  Buchner,  that  the  establishment  of  im- 
munity is  to  be  explained  by  reactive  changes  in  the 
integral  cells  of  the  body,  receives  additional  support, 
and  when  we  consider  the  observations  of  Bitter,2  who 
found  that  in  protective  vaccinations  against  anthrax 
the  vaccines  do  not  disseminate  themselves  through 
the  body,  as  is  the  case  when  the  virulent  organisms 
are  introduced,  but  remain  at  the  site  of  inoculation, 
and  from  this  point  produce,  by  the  absorption  of  their 
chemical  products,  the  systemic  changes  through  which 
the  animal  is  protected  against  subsequent  infection  by 
the  virulent  organisms,  we  feel  justified  in  concluding 
that  the  weight  of  evidence  is  strongly  in  favor  of  this 
view. 

1  Emmerich  and  Fowitzky:  Miinchener  med.  Wochensehrift,  1891, 
No.  32. 

2  Bitter :  Zeitschrift  fur  Hygiene,  1887,  Bd.  iv. 


6  00  HA  CTER  TO  LOG  Y. 

EHRLICH'S  "  SIDE-CHAIN  "  THEORY. — The  most  re- 
cent interpretation  of  the  phenomenon  of  acquired  im- 
munity is  that  proposed  by  Ehrlich  in  1897. ]  It  is 
generally  known  as  his  "  side  chain  "  or  "  lateral  bond  " 
(Seitenketten)  theory  of  immunity.  It  is  one  of  the  most 
attractive  of  all  the  hypotheses  that  have  been  advanced, 
and  is  in  many  ways  the  most  satisfactory. 

Its  fundamental  features  comprise  the  acceptance  of 
Weigert' s  doctrine  concerning  the  mechanism  of  physio- 
logical tissue-equilibrium  and  repair;  and  the  assump- 
tion of  a  specific  combining  relation,  or  affinity,  between 
toxic  substances  and  the  cells  of  particular  tissues. 

At  the  meeting  of  German  Naturalists  and  Physicians 
held  at  Frankfort-on-the-Main,  in  1896,  Weigert2  ad- 
vanced an  hypothesis  the  essential  features  of  which 
are  that  physiological  structure  and  function  depend 
upon  the  equilibrium  of  the  tissues  maintained  by  virtue 
of  mutual  restraint  between  its  component  cells  ;  that  de- 
struction of  a  single  integer  or  group  of  integers  of  a  tissue 
or  a  cell  removes  a  corresponding  amount  of  restraint  at 
the  point  injured,  and  therefore  destroys  equilibrium  and 
permits  of  the  abnormal  exhibition  of  bioplastic  ener- 
gies on  the  part  of  the  remaining  uninjured  components, 
which  activity  may  be  viewed  as  a  compensating  hyper- 
plasia ;  that  hyperplasia  is  not  therefore  the  direct 
result  of  external  irritation,  and  cannot  be,  since  the 
action  of  the  irritant  is  destructive  and  is  confined  to 
the  cells  or  integers  of  cells  that  it  destroys,  but  occurs 
rather  indirectly  as  a  function  of  the  surrounding  unin- 
jured tissues  that  have  been  excited  to  bioplastic  activity 

1  Ehrlich:  Klinisches  Jahrbuch,  1897,  Bd.  vi.  Heft  2,  S.  309. 

2  Weigert,  Carl :  "  Neue  Fragestellungen  in  der  pathologischen  Anat- 
omic," Verhandlungen  der  Ges.  deutscher  Naturforscher  und  Aerzte, 
1896,  S,  121. 


INFECTION  AND  IMMUNITY.  601 

through  the  removal  of  the  restraint  hitherto  exerted  by 
the  cells  destroyed  by  the  irritant ;  and,  finally,  when 
such  bioplastic  activity  is  called  into  play  there  is  always 
A^ercompensation — i.  e.,  there  is  more  plastic  material 
generated  than  is  necessary  to  compensate  for  the  loss. 
Ehrlich  applies  this  idea  to  the  individual  cell,  which 
he  conceives  to  be  a  complex  molecule,  comprising  a 
primary  central  nucleus  to  which  are  attached  by  side 
chains  its  secondary  atom-groups,  in  much  the  same  way 
that  our  conception  of  the  reaction-structure  of  complex 
organic  chemical  compounds  is  represented  graphically. 
Injury  to  one  or  more  of  these  physiologically  essential 
atom-groups  results,  according  to  the  view  of  Weigert, 
in  disturbance  of  the  cell-equilibrium  and  consequent 
eifort  on  the  part  of  the  surrounding  atom-groups  at 
compensatory  repair.  With  this  liberation  of  bioplastic 
energy  there  is  more  plastic  material  generated  than  is 
necessary  for  the  repair  of  the  injury.  The  excess  of 
this  material  finds  its  way  into  the  blood  and,  as  we 
shall  see  presently,  is  regarded  by  Ehrlich  as  the  real 
antitoxic  substance.  Assuming  a  specific  combining 
relation  between  toxic  substances  and  particular  cells  or 
secondary  atom-groups  of  cells — and  there  are  experi- 
mental grounds  for  this  assumption1 — it  is  evident  that 
the  combination  between  the  intoxicant  and  the  partic- 
ular atom-group  for  which  it  has  a  specific  affinity  is 
indirectly  the  cause  of  compensatory  bioplastic  activity 
on  the  part  of  similar  surrounding  atom-groups  that 
have  not  been  destroyed.  This  results,  as  we  learned 

1  See  Wassermann  und  Takaki :  "  Ueber  tetanus  antitoxische  Eigen- 
schaften  des  norraalen  Central nervensystems,"  Berliner  klin.  Wochen- 
schrift,  1898.  No.  1.  S.  5.  Neisser  und  Wechsberg:  Zeitschrift  fur 
Hygiene  und  Infektionskrankheiten,  Bd.  xxxvi.  S.  299.  Madsen : 
Ibid.,  Bd.  xxxii.  S.  214. 


602  BACTERIOLOGY. 

above,  in  hypercompensation,  the  excess  of  plastic  mate- 
rial being  disengaged  from  the  parent-cell  and  thrown 
free  into  the  circulating  fluids,  there  to  combine  directly 
with  the  same  intoxicant  should  it  subsequently  gain 
access  to  the  animal.  This  excess  of  plastic  material 
thrown  into  the  circulation  combines,  according  to  P^hr- 
lich,1  directly  with  the  intoxicant  to  form  physiologic- 
ally inactive  toxin — antitoxin  compounds  (see  page  497), 
and  can  therefore  be  reasonably  regarded  as  the  antitoxic 
material  of  animals  immune  from  bacterial  and  other 
toxins.  Since  the  announcement  of  that  doctrine  many 
important  advances  have  been  made  in  our  knowledge  of 
the  subject.  AVe  have  learned  that  immunity  or  tolerance 
may  be  induced  by  the  use  of  other  intoxicants  than  those 
elaborated  by  bacteria,  and  by  the  employment  of  other 
cells  and  cell  secretions.  It  has  been  demonstrated  that 
anti-bodies,  differing  in  their  specific  actions  from  anti- 
toxins, but  originating  probably  in  a  similar  manner,  are 
to  be  detected  in  the  fluids  of  animals  thus  immunized  or 
rendered  tolerant.  For  a  long  time  we  have  known  of 
the  germicidal  action  of  normal  blood-serum  ;  for  several 
years  we  have  been  familiar  with  the  singular  bacterio- 
lytic  phenomenon  demonstrated  by  Pfeiffer  in  the  peri- 
toneum of  animals  immune  of  cholera ;  more  recently 
we  have  learned  that  immunity  from  a  variety  of  infec- 
tions is  accompanied  by  a  power  on  the  part  of  the 
serum  of  the  immune  animal  to  agglutinate  the  bacteria 
causing  the  infection ;  and  the  profoundly  interesting 
investigations  of  Bordet,  Moxter,  von  Dungern,  Fish, 
and  others,  have  shown  that  immunity  may  be  induced 
from  cells  and  secretions  of  animal  origin  hitherto  re- 

1  Ehrlich  :  "  Zur  Kenntniss  der  Antitoxinwirkting,"  Fortschritte  der 
Medicin,  1897,  Bd.  xv.  No.  2. 


INFECTION  AND  IMMUNITY.  603 

garded  as  non-irritating  and  harmless.  For  instance, 
we  have  learned  that  the  blood  of  one  animal  may  cause 
fatal  intoxication  when  injected  into  an  animal  of  dif- 
ferent species;  but  if  that  blood  be  repeatedly  injected 
in  non-fatal  amounts,  the  animal  receiving  the  injections 
after  a  while  becomes  tolerant,  and  its  serum  reveals  the 
property  not  only  of  robbing  the  alien  blood  of  its 
hurtful  properties,  but  also  of  actually  dissolving  its 
corpuscles  (ha?molysis)  in  a  test-tube.  In  an  analogous 
way,  if  such  tissue-cells  as  epithelium  or  spermatozoa 
be  injected  repeatedly  into  the  tissues  of  animals,  the 
serum  of  the  blood  of  those  animals  acquires  the  power 
of  dissolving  (digesting)  such  cells  outside  the  body ; 
and  if  so  inert  a  secretion  as  milk  be  injected  into  the 
tissues,  the  blood-serum  of  the  animal  receiving  the 
injections  after  a  time  reacts  specifically  with  that  milk 
in  a  test-tube — L  e.,  precipitates  it.  From  the  foregoing 
we  see  that  in  the  numerous  phases  and  expressions  of 
this  physiological  possibility  there  are  produced  anti- 
bodies having  functions  totally  different  from  those 
attributed  by  Ehrlich  to  antitoxins — i.  e.,  we  have 
lysins,  agglutinins,  precipitins,  etc.,  that  in  their  mode 
of  action  suggest  ferments  with  specific  affinities.  It  is 
evident  that  when  broadly  conceived  the  mechanism  of 
immunity  comprehends  very  much  more  than  the  neu- 
tralization of  a  bacterial  toxin  by  an  antitoxin;  and, 
what  is  more  to  the  point,  in  many  of  these  conditions 
Gi  immunity  or  tolerance  above  noted  antitoxins  as  we 
know  them,  are  not  present  at  all. 

In  an  important  series  of  papers  on  the  ha3molysins 
published   by   Ehrlich   and   Morgenroth1  an    effort   is 

1  Ehrlich  und  Morgenroth :  Berliner  klinische  Wochenschrift,  1899, 


604  BACTERIOLOGY. 

made  to  elucidate  further  the  finer  mechanism  of  im- 
munity in  its  broad  sense  and  various  expressions, 
and  to  adapt  the  side-chain  doctrine  to  those  more 
complicated  phenomena  in  which  immunity  depends  not 
only  on  the  elaboration  of  antitoxins,  but  also  upon  a 
power  on  the  part  of  the  animal  fluids  to  cause  a  com- 
plete metamorphosis  or  disappearance  of  such  participate 
matters  as  bacterial  and  other  irritating  or  poisonous 
cells  and  substances.  They  believe  the  forces  at  work 
in  the  establishment  of  immunity  from  bacteria  and 
from  bacterial  and  other  toxins,  those  operative  in  the 
elaboration  of  the  newly  discovered  lysins,  antilysins, 
agglutinins,  precipitins,  ferments,  antiferments,  etc.,  as 
well  as  those  concerned  in  physiological  assimilation  and 
nutrition,  to  be  fundamentally  identical.  They  believe 
susceptibility  in  general,  as  well  as  power  to  assimilate 
nutrition,  to  be  explainable  through  the  assumption  that 
special  molecular  groups  of  the  living  protoplasm  are 
endowed  with  specific  combining  affinities  for  particular 
matters;  and  in  so  far  as  the  establishment  of  disease  is 
concerned,  they  regard  the  receptivity  of  the  individual 
to  be  determined  entirely  by  the  greater  or  less  suscepti- 
bility of  those  protoplasmic  molecular  groups — "  recept- 
tors,"  as  they  designate  them — to  disease-producing 
agents.  In  individuals  that  have  been  artificially  im- 
munized from  hurtful  substances,  they  believe  (in  reit- 
eration of  Ehrlich's  view  expressed  above)  that  the 
receptive  molecules  have  been  more  or  less  multiplied, 
according  to  the  degree  of  immunity,  through  bioplastic 

Bd.  xxxvi.  S.  6  and  481 ;  1900,  Bd.  xxxvii.  S.  453  and  681;  1901,  Bd. 
xxxviii.  S.  251,  569,  598.  See  also  Schlussbetrachtung:  Ehrlich,  in 
Nothnasrel's  Speciellen  Pathologie  und  Therapie,  Bd.  viii.  Theil  i. 
Heft  3,  S.  161. 


INFECTION  AND  IMMUNITY.  605 

activity  of  similar,  unimpaired  atom-groups  surrounding 
those  more  directly  influenced  by  the  intoxicant  during 
the  process  of  immunization  (see  page  601);  and  that 
this  excess  of  such  "  receptors,"  although  physiologically 
useless,  being  of  no  known  service  to  normal  function, 
circulates  unchanged  in  the  blood,  and  serves,  through 
specific  combining  affinity  for  the  poison  against  which 
the  animal  has  been  rendered  immune,  to  protect  the 
normal  tissues  from  its  hurtful  action. 

According  to  the  nature  of  the  irritant  from  which 
the  animal  has  been  immunized,  the  "receptor"  is  con- 
ceived to  be  either  of  simple  or  complex  construction, 
and  its  protective  function  to  be  performed  in  either  a 
comparatively  simple  or  in  a  more  or  less  complicated 
and  roundabout  manner. 

As  a  result  of  his  studies  of  toxins,  Ehrlich  reached 
the  conclusion  that  they  are  composed  of  at  least  two 
functionally  distinct  atom-groups  :  the  one,  a  "  hapto- 
phore  "  group,  characterized  by  its  combining  tendencies  ; 
the  other,  a  "toxophore"  group,  distinguished  for  its 
intoxicating  powers ;  and  that  for  the  exhibition  of  its 
hurtful  characteristics  a  toxin  molecule  needs  to  be  first 
anchored,  so  to  speak,  to  the  susceptible  tissue  by  the 
"  haptophore  "  group,  after  which  its  intoxicating  char- 
acteristics are  exhibited  by  the  "  toxophore  "  group.  He 
conceives  •  the  "  receptors  "  to  be  likewise  provided  with 
"  haptophore  "  groups  that  pair  with  the  corresponding 
"  haptophores "  of  the  poison  to  which  the  animal  .is 
susceptible  or  from  which  it  has  been  immunized.  Where 
immunization  has  been  induced  against  such  relatively 
simple  substances  as  toxins,  ferments,  and  certain  cell 
secretions,  the  "  receptors  "  and  their  functions  are  com- 
paratively simple — i.  e.,  the  single  haptophore  of  the 


606  BACTERIOLOG  Y. 

simple  receptor  pairs  with  that  of  the  intoxicant  and  a 
physiologically  inert  complex  results.  He  conceives 
antitoxins  to  be  simple  receptors  of  this  type,  and 
believes  the  neutralization  of  toxins  by  them  to  take 
place  in  this  manner.  On  the  other  hand,  if  the  im- 
munization of  an  animal  is  accompanied  by  an  acquired 
power  on  the  part  of  its  serum  to  disintegrate  bacteria, 
to  dissolve  alien  erythrocytes,  to  digest  such  cellular 
elements  as  epithelium  and  spermatozoa,  to  precipitate 
milk,  or  agglutinate  bacterial  or  blood-cells,  as  the 
studies  of  Pfeiffer,  Bordet,  von  Dungern,  Moxter,  Fish, 
Belfonte  and  Carbon,  Metchnikoff,  Gruber,  Durham, 
Widal,  and  others,  have  demonstrated,  then  the  process 
becomes  less  simple,  and  the  atomic  grouping  of  the 
receptive  molecule  is  correspondingly  more  complex. 
In  some  cases  the  receptor  is  provided  with  both  a  hap- 
tophore  and  a  ferment-like  (zymophore)  group ;  the 
function  of  the  former  being  to  combine  with  and  hold 
in  close  proximity  to  the  latter  the  albumin  molecule 
that  is  to  be  destroyed  or  assimilated  ;  in  this  way 
bringing  and  holding  the  albumin  molecule  directly 
under  the  influence  of  the  zymophore  group.  In  other 
cases  the  "  receptor  "  functions  symbolically,  so  to  speak, 
with  a  complementary  something  that  circulates  nor- 
mally in  the  blood,  the  so-called  "  complement "  of  Ehr- 
lich  and  Morgenroth.  Under  these  circumstances  the 
"  receptor  "  is  conceived  to  be  provided  with  two  "  hap- 
tophore  "  groups,  and  becomes  an  "  amboceptor,"  there- 
fore, the  one  haptophore  of  which  takes  up  and  fixes 
the  invading  bacteria,  tissue-cell,  or  albumin  molecule, 
while  the  other  pairs  with  the  corresponding  hap- 
tophore of  the  complement,  fixing  the  latter  in  close 
proximity  to  the  invading  body,  and  thereby  favoring 


INFECTION  AND  IMMUNITY.  607 

the  immediate  destructive  activity  of  its  "  zymotoxic  " 
group. 

It  is  interesting  to  note  in  connection  with  this 
hypothesis,  that  both  "  receptors  "  and  "  complements " 
are  present  in  normal  susceptible,  as  well  as  in  immune 
animals,  but  that  during  immunization  only  the  "  recep- 
tors "  are  multiplied  as  a  result  of  the  specific  stimula- 
tion necessary  to  the  establishment  of  immunity. 

THE  ORIGIN  OF  COMPLEMENT. — The  origin  of  com- 
plement is  a  question  that  is  still  unsolved.  Some 
investigators  are  inclined  to  believe  that  it  is  derived 
from  the  leucocytes.  This  is  the  opinion  of  Metchnikoif 
and  his  associates,  while  others  believe  that  it  is  derived 
from  other  cells  and  organs  as  well  as  from  the  leucocytes. 
Again  other  investigators  believe  that  it  is  not  derived 
from  the  leucocytes  at  all,  but  from  certain  other  organ 
cells,  for  instance,  the  spleen,  pancreas,  liver,  and  the 
bone  marrow.  It  is  impossible  with  the  knowledge  at 
hand  at  the  present  time  to  state  definitely  the  origin  of 
the  complement. 

Multiplicity  of  Complement — Ehrlich  and  his  associates 
have  demonstrated  that  in  normal  serum  several  comple- 
ments occur  in  association.  These  complements  appar- 
ently have  somewhat  different  functions,  as  indicated  by 
the  influence  upon  blood  serum  when  saturated  with  cer- 
tain elements ;  in  this  way  removing  one  form  of  com- 
plement, and  leaving  the  others  intact.  Again,  filtering 
the  serum  through  a  porcelain  filter,  serves  to  separate 
some  complements  and  allows  others  to  remain  in  the 
serum. 

CONCLUSIONS. — According  to  the  nature  of  the  intoxi- 
cant from  which  the  animal  is  immunized,  the  one  or  the 
other  of  the  structurally  and  functionally  different  types 


608  BACTERIOLOGY. 

of  receptors  is  increased — i.  e.,  in  immunity  from  a  simple 
toxin  the  simplest  type  of  receptor  appears  in  the  blood 
(receptors  of  the  first  order,  Ehrlich) ;  in  immunity  that  is 
associated  with  agglutinating  or  precipitating  powers  on 
the  part  of  the  blood-serum  receptors  having  a  haptophore 
and  a  zymophore  group  appear  (receptors  of  the  second 
order) ;  while  in  immunity  from  such  molecular  com- 
plexes as  blood-,  tissue-,  or  bacterial  cells  there  are 
produced  receptors  of  the  third  order,  which  act  through 
their  haptophore  groups  as  intermediate  links  between  the 
body  to  be  destroyed  and  the  normally  present  ferment- 
like  complement  that  is  to  bring  about  the  destruction. 
For  all  the  foreign  irritants  from  which  animals  have 
been  immunized,  be  it  alien  blood,  tissue-cells,  milk,  or 
bacteria,  there  is  assumed  to  be  circulating  normally  in 
the  blood  a  "  complement "  specifically  related  to  that 
irritant  on  the  one  hand,  and  to  its  "  receptor "  on  the 
other.  This  idea  of  plurality  for  the  complement  is 
apparently  the  vulnerable  point  in  the  argument.  At 
all  events,  it  has  been  vigorously  assailed  by  Bordet  and 
Buchner,  especially,  who  consider  the  complement  as  a 
unit,  and  who  do  not  regard  it  as  possessed  necessarily 
of  specific  affinities  beyond  those  common  to  what  may 
be  termed  proteolytic  enzymes  in  general ;  and  Buchner 
regards  it  as  nothing  more  than  the  normally  present 
"  alexin,"  to  which  he  called  attention  years  ago. 
Whether  these  objections  be  well  taken  or  not,  whether 
the  doctrine  as  a  whole  can  be  accepted  or  not,  the  experi- 
mental data  on  which  it  is  based  warrant  the  opinion 
that  it  is  the  only  satisfactory  working  hypothesis  that 
has  been  offered  in  explanation  of  the  mechanism  of 
what  Buchner  years  ago  designated  the  "reactive  tis- 


INFECTION  AND  IMMUNITY.  609 

sue-changes  "  underlying  the  establishment  of  acquired 
immunity.1 

The  observations  serving  as  the  basis  for  this  doctrine 
have  given  to  the  blood  and  fluids  of  the  body  a  new 
and  peculiar  interest.  According  to  circumstances,  there 
may  be  detected  in  the  blood  and  tissue-juices  a  number 
of  bodies  having  totally  different  functions  and  affinities, 
and  therefore  presumably  different  from  one  another. 
To  summarize  briefly :  First,  there  is  normally  present 
in  the  blood-serum  of  practically  all  animals  the  de- 
fensive "alexins"  already  mentioned.  Second,  the 
antitoxins  that  are  found  in  the  blood  of  animals  arti- 
ficially immunized  from  special  sorts  of  infection  and 
intoxication,  as  well  as  occasionally  in  the  blood  and 
tissues  of  normal  animals,  the  functions  of  which  are 
susceptible  of  demonstration  outside  the  body  as  well  as 
within  the  tissues  of  the  living  animal.  Third,  a  body 
possessed  of  disintegrating,  bacteriolytic  powers,  a  bac- 
teriolysin — /.  r.,  having  the  property  of  actually  dissolving 
bacteria,  so  that  the  phenomenon  may  be  observed  under 
the  microscope.  This  phenomenon  is  especially  to  be  seen 
within  the  peritoneum  of  guinea-pigs  that  have  been  ren- 
dered immune  from  Asiatic  cholera  and  from  the  typhoid 
and  colon  infections  or  intoxications.2  It  is  not  to  be 
confounded  with  the  ordinary  bactericidal  function  of 
the  alexins  that  is  demonstrable  in  most  normal  serums. 
Fourth,  a  body,  the  so-called  "  agglutinin "  (Gruber), 
that,  was  considered  by  Widal  to  represent  a  "  reaction 
of  infection/7  and  not  of  immunity ;  though  at  this  time 
its  presence  is  generally  supposed  to  indicate  an  effort 

1  Justice  cannot  be  done  to  the  beauty  and  ingenuity  of  this  con- 
ception in  so  brief  a  summary  as  is  appropriate  to  a  text- book.    To  be 
appreciated  it  must  be  read  as  it  came  from  its  authors. 

2  It  is  generally  known  as  Pfeiffer's  phenomenon, 

39 


610  BACTERIOLOGY. 

on  the  part  of  the  body  to  resist  infection.  The  pres- 
ence of  this  body  in  a  serum  of  an  animal  is  announced 
by  its  peculiar  influence  on  the  activity  and  arrangement 
of  the  particular  species  of  bacteria  from  which  the  indi- 
vidual is  immune,  or  with  which  it  is  infected.  In  the 
case  of  typhoid  fever  in  man,  for  instance,  the  serum 
obtained  during  the  early  and  middle  stages  of  the  dis- 
ease, when  mixed  with  fluid  cultures  or  suspensions  of 
the  typhoid  bacillus,  causes  the  bacilli  to  lose  their 
motility  and  to  congregate  (agglutinate)  in  masses  and 
clumps,  a  condition  never  seen  in  normal  cultures  of  this 
organism,  and  practically  never  observed  when  normal 
serum  is  employed.  There  are  evidences  of  the  pres- 
ence of  "  agglutinin  "  in  certain  of  the  antitoxic  serums 
from  artificially  immunized  animals,  viz.,  that  of  ani- 
mals immune  from  cholera,  pyocyaneus,  typhoid,  dysen- 
tery, and  colon  infections.  So  far  as  experience  has 
gone,  this  agglutinating  property  is  manifested  in  the 
great  majority  of  cases  only  upon  the  particular  organ- 
isms from  which  the  animal  supplying  the  serum  is 
protected ;  that  is  to  say,  the  relation  is  specific.  In 
view  of  the  fact  that  the  power  of  a  serum  to  agglutinate 
bacteria  is  regarded  by  many  as  a  concomitant  of  infec- 
tion, the  exhibition  of  this  property  by  the  blood  of 
immune  animals  may  at  first  sight  appear  paradoxical. 
We  should  not  lose  sight  of  the  fact,  however,  that 
agglutinin  is  presumably  distinct  from  the  other  sub- 
stances concerned  in  immunity,  and  its  presence  in  im- 
mune animals  may,  therefore,  be  reasonably  explained 
as  a  more  or  less  permanent  result  of  the  "  reactions  of 
infection  "  that  were  coincident  with  the  primary  stimu- 
lations by  specific  infective  or  intoxicating  matters  nec- 
essary to  the  establishment  of  the  condition  of  immunity; 


INFECTION  AND  IMMUNITY.  611 

nor  should  we  in  this  connection  lose  sight  of  the  fact 
that  its  presence  is  constantly  to  be  demonstrated  in 
typical  cases  of  typhoid  fever,  for  instance,  that  termi- 
nate fatally,  and  that  have  exhibited  little  or  no  clinical 
signs  of  resistance  at  any  time  during  their  course. 
Fifth,  there  may  be  demonstrated  in  the  blood  of  ani- 
mals that  have  received  repeated  subcutaneous  injections 
of  milk  a  body — a  "  precipitin  " — that  causes  a  precipi- 
tation of  milk.  This  precipitation  represents  apparently 
a  specific  reaction,  for  it  occurs  only  when  the  blood- 
serum  is  mixed  with  milk  from  the  species  of  animal 
that  supplied  the  milk  used  for  the  injections.  Sixth, 
after  the  repeated  injection  of  blood  or  of  emulsions  of 
tissue-cells  into  the  body  of  an  animal,  there  appear  in 
the  blood  of  that  animal  certain  solvents,  or  enzyme-like 
bodies,  "  hsemolysins,"  "  cytolysins,"  etc.,  that  react 
specifically  upon  the  blood  or  tissue-cells  injected ; 
agglutinating,  disintegrating,  and  finally  completely 
dissolving  them.  Here,  too,  the  relations  are  specific. 
If  a  rabbit,  for  instance,  be  rendered  tolerant  to  or 
immune  from  beef-blood,  its  serum  dissolves  only  the 
red  corpuscles  of  bovines ;  if  from  dog's  blood,  then 
only  the  corpuscles  of  the  dog  are  dissolved  by  the 
serum  of  the  rabbit.  Similarly,  if  a  rabbit  be  rendered 
tolerant  to  injections  of  emulsions  of  epithelium  cells, 
then  its  serum  dissolves  epithelium  and  not  other  cells. 
All  these  reactions  may  be  seen  in  a  test-tube  or  under 
the  microscope.  Seventh,  if  a  hsemolyzing  serum,  pre- 
pared as  indicated  under  the  sixth  observation,  be  heated 
for  a  short  time  to  54°— 56°  C.,  it  at  once  loses  the 
hsemolytic  function,  but  regains  it  again  if  a  few  drops 
of  serum  from  a  normal  animal  be  added  to  it.  In  this 
phenomenon  of  haemolysis  Ehrlich's  "  receptors  of  the 


HIM  It  If'/ 

lllllll    Mllll'l    "    f|H>    MNHllMH'll    Itl    IK      Ml||M>HH>f|  i     |||t-    llHllillfi 

iliMlhivM  Hit  "  Hiin|ili>!iiHi!  /'  Hiiil  lIu'M-hv  II|IMII|M  iln  jnn. 

I'MH  I    Illll    ||||l    Ml|llNII||l|H|ll     Illldlllilll     III     (In      IHll'llltll     'Ml  Mill 

MII|I|I||HM  lVi»nli  "  i>Mlii|ilt'liiii|il("  Mini  til  MUM-  I.>|MH<  il,, 
•  iilhlh  nl  (lit-  liihliiiiK  f!ii|i  IIM|I|H|I  Kljililli,  II  lilimil 
l'llllllllll!ll|l  (I  llMMIHlh  "III  III  M  i'Unl\"lll  lid  hi|!li||||i||lv 

ln|i'i'h'il  lulu  iiiniiiliiiiil,  null  liHilli'M     "  iiiillly«"liNn     -m 

I'll  Illl'll,  Illlll    I  lll>  H|I|  Din   ill    llii-  iilillii'll     li-i      tin      jintn  i    ill 
IlllllllllfUl   llHMIHlh  III'    MMlim   nl    II'     Ilii'llinh  flll)i     Illlii  Iliiii 
II    ml-..  .1    \Sllll   II    III  M  It     I    lul"    '        Nllilli,  II    Mill  Illlll  III.  .,  ..I 
l»ti||||l|l  (III  III  ,    III'     ll||i'i-|iil     Illlii    llii>    M,inn     Ml 
n|H'i-li»«    III     (Mlllllfll,    illllli'iilll|l!t'llll'lll     lit 


1    II     I"    i   .1.1.  Ill     hulll     Mli.il     Ilil'l    III  I'M      .H.l     Hi  .1    H,,     |||.|||<I    I*.   .,    .  M.,1 

ii.  iiiii.  i.  I'll  (Mill-Hull  |i,'.'-'i  '-M  .1  IM    lln>  Hut.  I"  .mil  HHHMKH  nl   HM*  lituh 

I"    Wlll»'H|M»'llll  |     l«     lllHI'll     MjIMM     MM*    I"   'I    Ml     t'»|U'l  iMM'MlllI     .  »  I.I.  n.  , 
Illlll    lhlHMi.in.il    .(.*   I  hi'  HllttllMM  |»»l»ll    lui     .ill     (In     lMi|ini|.i(il     Inn   -II 
hUlliMIrt    HlKl    IliMf    lit'i  II    JMHhMMM'Mlill    III    lUMllhliltH    "HI    |MI>MlMll   lili  -I  - 
III    IIMIMMMlh         Nlll\\  UllHlllMlIlM^     !I»K  HIM'    tmiHllMlldh     ..III  nilllli  i  '   •» 
•  ll»«mli  »      l».uiuii..»Hi  n    In  .in  -nlilii  •>•«  In  Inn.  ih.   Hi  nu.tii  I'nlllulunll'Hl 

»«UI-h.h    III    Mllllll'll   !"•'••    l»>  llllUI    MlMl'llll.    \\  Ml  III.  U'l  (Mill     l"llll,    N.I     II  • 
ML  1.  1.    -i    i  li,.iinin  .iM>ii-l>    M|inli  H»i-  i  i  lili  Mil-  llnl    ll'l  •    l«<  •  H    p»»--inlt.l    III 
lUnn  nl    .»  y|||\|   pVUHll'Mltl    iMMt'lluM  ill    HM>   I0*u»il  "I'HIIU        lit'  ln.ll.  vi  « 
llM1  »|»'rt|MM'lluM  Ml   liiM'li'llil  nlci  inil   \\  \\\  \\  Hi.  »    in    nil  %.  il  nllli  lilnntl 
<\\\\\\\  lit  \W  I!MI>  li'.'H  |M  Vllitl  \\\\\\\  lu  |iln-<lnil  I..IM  .1  ••      Id    ».  ,,.inl  -  1  1.. 

\\><\\\\  u|    llu-  um.»MlMM'«  ih  IU"ll  "I'Mtin  .(  «    I  In.   Ii    .nil   Ml    ill  .Mnl«-»u.  .    •  Ml 

\\\\\\    -<\\\\\\\\\    h.in-'l\  n  n>  i> 


\\\\*\\\\\\\\  Ml 

i  MUl  »»»»!  .H  n  h'-nnl  M| 

H»i    IUttlVtilMU|M  Ml  llu<  "I'MMM 

M|\»\U    llU'    (Mn-Mln-nltUn    ntutnl  HM   In   III.    l-il-,« 
W  \\\\\\  \\\   \\nl-  »M  r.'».i  t*w    AvIu'lUMt  MUM  »h»»« 
IVU\\IM^  M,  1^1    I   \\\\\\  III  \ 
\\    \\\*\    \\\-    \\\\>  il,>i  »UM.      i  ,  -MI  Hi.,    rt*   II    ^M» 

»»•'"»     W\     ill  -MM,,MnlMa      -U\     l\\\M\\Mll\l      IMM-M     »«i      ,,tn  M     .III.       .  --M    l.».   ,   , 

M^\M      A«  \(>|    II  l\»^  MM!  »\H^M'lvil   yvn   ^^M^H!  ^MvUll\\Ui   \\\\\   \\\\\ 

u  i«  M\l  |MtoWlW\!^\  HHMI  H^»  ^\i»^u»^  ^I\^M\>^\\  Ivy 

l^\"  ^WM   .««l\|»>».|v»\  tv*  \'.uv\\\\  t>«^'Umt'M^O   -v\y\MM\  Isy 

\\  \\\\n\\w\\m    \\\\\\\  *\\v\\  m^MHws  \\\\\ 


/  \  /  /                        I   \  /'     /  I/  IM     \  /  M  III    I 

•  I     1 1 1 1  •  1 1  •  1 1 1 1 1 .     !  I 


I  I,     i    M'U'lllllH  *,(,,  |{  I.  HtttllHl     I | I    •      < 

tli     .  <  i  -I  |M!MM  'i«-i«  !••»   i ••!  * ..unnm^  in 

1  |"  '"''  -I    "I Mil.-    ill .,,,       ,    ,  , 

<  M    i«'-"  ••'••Ih      ,     i i,  , 

•  •mm.  •!    I. MI   il l.M,         ,      ,11 ||m  Hi,,,,, 

•'     II  III    '•      '"••  ' '       ''•         I"    llill  III      llM'i     I 

MI-      II    '      Aw  w  hmiill   til1  Biicli   liivi<»liniilliiiin 

1  ••       Iwl       "i    '     ' '  i  "-Will 

1        i    .     i    M»WM  I 

I      N '     l>|  '          ••   •  "    •     i 

II  ' i    IM  I  MM.  I  Itttfliii*,  •    UtlUMl     '     ••  •'•     r  •" 

-I   ii-     '  '      ••    \\\\    f|j   HM    |MiUniiniiM  (ihuliioU  nC 

(l •     •••  '    •     'iii      id     i  .«<  •    tin  in  i.   till 

i     ||||  -             til      M|l|U    Hlfl     II..      |..     i,ll..  ||{ 
111  •"       III  III   '         || |l  •   "ll   I'dlU, 

1  '•  il    ' in    'i  nmy  I       i 

I  Idlli  '    II     (l i      Ij   ••    -I   n,,    i..  i   .,  ,i   ,  ,    ||Mi|M 

''"        .',!,, I    ,1,1      ,,,      |||i(|(|  ,   I,      ,       ,,,,,(,   ,,|          ,,,    ,,  I I      I. 

(l" MIIIIMK  Illlll    llllW  li.«-  •'    -      I       -'I.    il, 

I  ItMilvIl         '    ll(1     |'l"",",'     II       ,.,  I    ,  •      ||| 

••I  HIM  i '    ' 

1   '•  \\     III    I' I     'I I       ||     |||  .Hit.,    IJ I 

1    III  III  •'     |L  •      Pftlftl      •  ><iiliHlinti .    H. 

jHihl       II|H|I|       i    H.     i     i        ,    ,   ,.  i  . 

I    [\\\          III        I     •     '     •  •  •      I   ||{          |||    !,,!,.               ,       ,,, 
II     ' "      I ||  ,       I        I          ||| ,,!,,!, 

I •   l-lnii    I  ('I      i,i,  .1.      ,,,  ,  ,,,i    , 

i      i    ,,,  M.  i    ,i,    ,  i 
i,,  i,. i 

i     1 1.  ,i                                 i,    ,.,  ,,ii  , . ,,  .     i  ,M 

11          ''     i  '•"  IM  Ml 


614  BACTERIOLOGY. 

of  bacteria  that  in  some  way  or  other  have  been 
modified.  This  mollification  may  be  artificially  pro- 
duced in  the  products  of  virulent  organisms,  and 
then  introduced  into  the  tissues  of  the  animal ;  or  the 
virulent  bacteria  may  be  so  treated  that  they  are  no 
longer  of  full  virulence,  and  when  introduced  into 
the  body  of  the  animal  will  produce  poisons  of  a 
much  less  vigorous  nature  than  would  otherwise  be  the 
case. 

5.  That  immunity  following  the  introduction  of  bac- 
terial products  into  the  tissues  is  apparently  due  to  the 
formation  in  the  tissues  of  another  body  or  other  bodies 
that  act  as  antidotes  to  the  poisons,  and  thereby  protect 
the  tissues  from  their  hurtful  effects. 

6.  That  this  protecting  proteid  which  is  generated  by 
the  cells  of  the  tissues  need  not  of  necessity  be  antago- 
nistic to  the  life  of  the  invading  organisms  themselves, 
but  in   most  cases  must  be  looked   upon  more  as  an 
antidote  to  their  poisonous  products. 

7.  That  immunity,  as  conceived  by  Ehrlich,  may  be 
either  "  active  "  or  "  passive."     According  to  this  inter- 
pretation, it  is  "  active "  when  resulting  from  an  ordi- 
nary non-fatal  attack  of  infectious  disease;  or  from  a 
mild  attack  of  infection  purposely  induced  through  the 
use  of  living  vaccines ;   or  from  the  introduction   of 
cultures  of  the  bacteria  that  have  been  killed  by  heat ; 
or  from  the  gradual  introduction  of  toxins  into  the  tis- 
sues until  a  marked  antitoxic  state  is  reached.     It  is 
'*  passive  "  when  occurring  as  a  result  of  the  direct  trans- 
ference of  the  perfected  immunizing  substance  from  an 
immune  to  a  susceptible  animal,  as  by  the  injection  of 
blood-serum  from  the  former  into  the  latter.     "  Passive 
immunity  "  is,  in  most  cases,  conferred  at  once,  without 


INFECTION  AND  IMMUNITY.  615 

the  delay  incidental  to  the  usual  modes  of  establishing 
"  active  immunity."  As  a  rule,  "  active  "  is  more  lasting 
than  "  passive  "  immunity. 

8.  That  phagocytosis,  though  frequently  observed,  is 
effective  in  warding  off  disease  in  normal  individuals 
only  when  the  normal  defenses  of  the  body  are  fully 
active ;  when  the  number  of  invading  bacteria  is  rela- 
tively small ;  or  when  the  bacteria  are  possessed  of  low 
aggressive  powers ;  while  in  acquired  immmunity  it  is 
more  probably  a  secondary  process,  the  bacteria  being 
taken    up   by  the    leucocytes   only  after   having   been 
modified   in   virulence  through  the  germicidal  activity 
of  the  serum  of  the  blood  and  of  other  fluids  in  the 
body.     It  is,  however,  probable  that  the  living  leuco- 
cytes contribute  to  the  circulating  fluids  certain  sub- 
stances that  are  important  to  the  establishment  of  im- 
munity. 

9.  That  of  the  hypotheses  advanced  in  explanation 
of  acquired  immunity,  the  one  worthy  of  greatest  con- 
fidence is  that  which   assumes  immunity  to  be  due  to 
reactive  changes  on  the  part  of  the  tissues  that  result 
in  the  formation  in  these  tissues  of  antitoxic  and  other 
anti-bodies,  which  circulate  free  in  the  blood,  and  in  a 
variety  of  ways  serve  to  protect  the  tissues  from  the 
harmful  effect  of  extraneous  intoxicants  and  irritants, 
in  some  cases  acting  principally  as  antidotes  to  a  toxin, 
in  others  exhibiting  more  the  germicidal  (bacteriolytic) 
than  the  antitoxic  property. 


CHAPTER    XXYI. 

Bacteriological  study  of  water — Methods  employed — Precautions  to  be 
observed — Apparatus  employed,  and  methods  of  using  it — Methods 
of  investigating  air  and  soil — Bacteriological  study  of  milk — 
Methods  employed. 

BACTERIOLOGICAL   STUDY    OF   WATER. 

THE  conditions  that  favor  the  epidemic  outbreak  of 
typhoid  fever,  Asiatic  cholera,  and  other  maladies  of 
which  these  may  be  taken  as  types,  have  served  as  a 
subject  for  discussion  by  sanitarians  for  a  long  time. 

Of  the  hypotheses  that  have  been  advanced  in  ex- 
planation of  the  existence  and  dissemination  of  these 
diseases,  two  stand  pre-eminent  and  are  worthy  of  con- 
sideration. They  are  the  "ground-water"  theory  of 
von  Pettenkofer  and  his  pupils,  and  the  "  drinking- 
water"  theory  of  the  school  of  bacteriologists  of  which 
Koch  stands  at  the  head. 

The  adherents  to  the  "ground-water"  view  explain 
the  occurrence  of  these  diseases  in  epidemic  form  through 
alterations  in  the  soil  resulting  from  fluctuations  in  the 
level  of  the  soil-water;  and  assign  to  drinking-water 
either  a  very  insignificant  role,  or,  as  is  most  frequently 
the  case,  ignore  it  entirely.  On  the  other  hand,  those 
who  have  been  instrumental  in  developing  the  drinking- 
water  hypothesis  claim  that  alterations  in  the  soil  play 
little  or  no  part  in  favoring  the  outbreak  of  these  dis- 
eases ;  but  that,  as  a  rule,  they  appear  as  a  result  of 
direct  infection,  through  the  use  of  waters  contaminated 

616 


BACTERIOLOGICAL  STUDY  OF  WATER.       617 

with  materials  containing  the  specific  organisms  known 
to  cause  such  diseases. 

As  a  result  of  numerous  observations  by  the  disciples 
of  both  schools,  the  evidence  is  now  greatly  in  favor  of  the 
opinion  that  polluted  water  is  primarily  the  underlying 
cause  of  these  epidemics,  and  this  too,  very  often,  when 
the  state  of  the  soil-water,  in  the  light  of  the  "  ground- 
water"  hypothesis,  is  just  the  reverse  of  what  it  should 
be  in  order  to  render  it  answerable  for  them.  It  is 
manifest,  therefore,  that  the  careful  bacteriological  study 
of  water  intended  for  domestic  use  is  of  the  greatest 
importance,  and  should  be  a  routine  procedure  in  all 
communities  receiving  their  Avater-supply  from  sources 
liable  to  pollution. 

The  object  aimed  at  in  such  investigations  should  be 
to  determine  the  number  and  kind  of  bacteria  con- 
stantly present  in  the  water — for  all  waters,  except 
deep  ground- water,  contain  bacteria ;  if  sudden  fluctua- 
tions in  the  number  and  kind  of  bacteria  occur  in 
these  waters,  and  if  so,  to  what  they  are  due ;  and 
finally,  and  most  important,  whether  the  water  contains 
constantly,  or  at  irregular  periods,  bacteria  that  can  be 
traced  to  human  excrement,  not  of  necessity  pathogenic 
varieties,  but  bacteria  that  are  known  to  be  present 
normally  in  the  intestinal  canal.  For  if  conditions 
arc  continuously  favorable  to  pollution  of  the  water 
by  the  normal  constituents  of  the  intestinal  canal,  the 
same  conditions  would  allow  of  the  occasional  pollution 
of  such  water  by  infective  matters  from  the  bowels  of 
persons  suffering  from  specific  disease  of  the  intestines. 

In  considering  water  from  a  bacteriological  stand- 
point it  must  always  be  borne  in  mind  that  com- 
parisons with  fixed  standards  are  not  of  much  value, 


618  BA  CTERIOL  OGY. 

for  just  as  normal  waters  from  different  sources  are 
SL'cn  to  present  variations  in  their  chemical  composition, 
without  being  unfit  for  use,  so  may  the  relative  number 
and  variety  of  species  of  bacteria  in  water  from  one 
source  be  always  greater  or  smaller  than  in  that  from 
another,  and  yet  no  difference  may  be  seen  to  result 
from  their  employment.  For  this  reason  systematic 
study  of  any  water,  from  this  point  of  view,  should 
begin  with  the  establishment  of  what  may  be  called  its 
normal  mean  number  of  bacteria,  as  well  as  the  charac- 
ter of  the  prevailing  species ;  and  in  order  to  do  this 
the  investigations  must  cover  a  long  period  of  time 
through  all  the  seasonal  variations  of  weather.  From 
data  obtained  in  this  way  it  may  be  possible  without 
analysis  to  predict  approximately  at  any  season  the 
bacteriological  condition  of  the  water  studied.  Marked 
deviations  from  these  "  means/7  either  in  the  quantity 
or  quality  of  the  organisms  present,  can  then  be  con- 
sidered as  indicative  of  the  existence  of  some  unusual, 
disturbing  element,  the  nature  of  which  should  be 
investigated.  It  is  impossible  to  formulate  an  opinion 
of  much  value  from  either  a  single  chemical  or  bac- 
teriological analysis  of  a  water,  or  from  both  together 
in  many  cases;  for  the  results  thus  obtained  indicate 
only  the  condition  of  the  water  at  the  time  the  sample 
was  procured,  and  give  no  indication  as  to  whether  it 
differed  at  that  time  from  its  usual  condition,  or  from 
the  normal  condition  of  the  waters  of  the  immediate 
neighborhood. 

.  The  interpretation  of  the  results  of  both  chemical 
and  bacteriological  analyses  of  a  sample  of  water  ac- 
quires its  full  value  only  through  comparison,  either 
with  "means"  that  have  been  determined  for  this 


BACTERIOLOGICAL  STUDY  OF   WATER.       619 

water,  or  with  the  results  of  simultaneous  analyses  of 
a  number  of  samples  from  other  sources  of  supply  of 
the  locality. 

The  aid  of  the  bacteriologist  is  frequently  sought  in 
connection  with  investigations  of  waters  that  are  sup- 
posed to  be  concerned  in  the  production  of  disease,  par- 
ticularly typhoid  fever,  either  in  isolated  cases  or  in 
widespread  epidemic  outbreaks,  and  in  these  cases  both 
the  bacteriologist  and  the  person  employing  his  services 
are  cautioned  against  being  too  sanguine  of  positive 
results,  for  in  the  vast  majority  of  instances  reliable 
bacteriologists  fail  to  detect  in  these  waters  the  bacillus 
that  is  the  cause  of  typhoid  fever. 

Failure  to  find  the  organism  of  typhoid  fever  in 
water  by  the  usual  methods  of  analysis  does  not  by 
any  means  prove  that  it  is  not  present  or  has  not 
been  present.  The  means  ordinarily  employed  in  the 
work  admit  of  such  a  very  small  volume  of  water  being 
used  in  the  test  that  we  can  readily  understand  how 
typhoid  bacilli  might  be  present  in  moderate  numbers 
and  yet  none  be  included  in  the  drop  or  two  of  the 
water  taken  for  study.  The  conditions  are  not  those 
of  a  solution,  each  drop  of  which  contains  exactly  as 
much  of  the  dissolved  material  as  do  all  other  drops  of 
equal  volume ;  but  are  rather  those  of  a  suspension,  in 
every  drop  or  volume  of  which  the  number  of  sus- 
pended particles  is  liable  to  the  greatest  degree  of 
variation.  Furthermore,  there  are  other  reasons  that 
would,  a  priori,  preclude  our  expecting  to  find  the 
typhoid  bacilli  in  water  in  which  we  may  have  reason 
to  believe  they  had  been  deposited,  because  attention  is 
not  usually  directed  to  the  water  until  the  disease 
has  become  conspicuous,  usually  in  from  two  to  four 


620  BACTERIOLOGY. 

weeks  after  the  pollution  probably  occurred.  These 
intervals  of  time  are  ordinarily  sufficient  for  the  deli- 
cate, non-resistant  bacillus  of  typhoid  fever  to  succumb 
to  the  unfavorable  conditions  under  which  it  finds  itself 
in  water.  By  unfavorable  conditions  are  meant  the 
absence  of  suitable  nutrition;  unfavorable  temperature; 
probably  the  antagonistic  influence  of  more  hardy 
saprophytic  bacteria,  particularly  the  so-called  "  water- 
bacteria/7  and  of  more  highly  organized  water-plants ; 
the  effect  of  precipitation  and  of  sedimentation  ;  and, 
of  great  importance,  the  disinfecting  action  of  direct 
sunlight. 

Though  the  positive  demonstration  of  typhoid  bacilli 
in  drinking-water  by  bacteriological  methods  is  of  ex- 
treme rarity,  it  must  not  be  concluded  that  bacteriological 
analyses  of  suspicious  waters  shed  no  light  upon  the  exist- 
ence of  pollution  and  the  suitability  or  non-suitability 
of  the  water  for  drinking-purposes. 

In  the  normal  intestinal  tract  of  all  human  beings 
and  of  many  other  mammals,  as  well  as  associated  with 
the  specific  disease-producing  bacillus  in  the  intes- 
tines of  typhoid-fever  patients,  is  an  organism  that  is 
frequently  found  in  polluted  drinking-waters,  and 
whose  presence  is  proof  positive  of  pollution  by  either 
normal  or  diseased  intestinal  contents ;  and  though 
efforts  may  result  in  failure  to  detect  the  specific 
bacillus  of  typhoid  fever,  the  finding  of  the  other 
organism,  bacillus  coli,  justifies  one  in  expressing  the 
opinion  that  the  water  under  consideration  has  been 
polluted  by  intestinal  evacuations  from  either  human 
beings  or  animals.  Waters  so  exposed  as  to  be  liable  to 
such  pollution  should  never  be  considered  as  other  than 
a  continuous  source  of  danger  to  those  using  them. 


BACTERIOLOGICAL  STUDY  OF   WATER.       621 

Another  point  to  be  remembered  is  in  connection  with 
chlorine  as  an  indicator  of  contamination  by  human 
excrement.  It  is  commonly  taught  that  an  excessive 
amount  of  chlorine  in  water  points  to  contamination  by 
human  excreta.  This  may  or  may  not  be  true,  accord- 
ing to  circumstances.  A  high  proportion  of  this  element 
in  a  sample  of  water  from  a  locality,  the  surrounding 
waters  of  which  are  poor  in  chlorine,  is  unquestionably 
a  suspicious  indication ;  but  in  a  district  close  to  the  sea 
or  near  salt-deposits,  for  instance,  where  the  proportion 
of  chlorine  (as  chlorides)  in  the  water  is  generally 
high,  the  value  of  the  indications  thus  afforded  is  very 
much  diminished  unless  the  amount  found  in  the  sample 

»  under  examination  greatly  exceeds  the  normal  "  mean," 
previously  determined,  for  the  amount  of  chlorine  in 
the  waters  of  the  neighborhood. 

A  striking  example  of  the  latter  condition  occurred 
in  the  experience  of  the  writer  while  inspecting  a 
group  of  water-supplies  on  the  east  coast  of  Florida. 
In  each  instance  the  water  was  obtained  from  properly 
protected  artesian  wells,  ranging  from  200  to  400 
feet  deep,  and  located  within  a  few  hundred  yards 
of  the  sea.  The  first  sample  subjected  to  chemical 
analysis  revealed  such  an  unusually  high  proportion 
of  chlorine  that,  had  this  sample  alone  been  con- 
sidered, the  opinion  that  it  was  polluted  by  human 
excreta  might  have  been  advanced.  To  prevent  such 
an  error  samples  of  water  from  a  number  of  wells  in 
the  neighborhood  were  examined,  and  they  were  all 
found  to  contain  from  ten  to  twelve  times  the  amount 
of  chlorine  that  ordinarily  appears  in  inland  waters,  the 
excess  being  evidently  due  to  leakage  through  the  soil 
into  the  wells  of  water  from  the  sea.  In  short,  the 


622  BA  CTERIOLOG  Y. 

presence  of  an  excess  of  chlorine  in  water,  while  often 
indicating  pollution  from  human  evacuations,  may 
nevertheless,  sometimes  arise  from  other  sources ;  but 
the  presence  in  water  of  bacteria  normally  found  in  the 
intestinal  canal  can  manifestly  admit  of  but  one  inter- 
pretation, viz.,  that  ftecal  matters  from  either  man  or 
animals  have  at  some  time  been  deposited  in  this  water, 
and  that  while  no  specific  disease-producing  organisms 
may  have  been  detected,  still  waters  in  which  such  pol- 
lutions are  possible  are  a  constant  menace  to  the  health 
of  those  who  use  them  for  domestic  purposes. 

A  sudden  variation  from  the  normal,  mean  number 
of  bacteria,  or  from  the  normal  chemical  composi- 
tion of  a  water,  calls  at  once  for  a  thorough  in- 
spection of  the  supply,  while  at  the  same  time  the 
organisms  present  are  to  be  subjected  to  the  most  care- 
ful study.  In  many  instances,  even  after  the  most 
thorough  bacteriological  and  chemical  study  of  a  sus- 
picious water,  one  is  forced  to  admit  that  information 
of  but  limited  usefulness  has  been  obtained  through  the 
employment  of  such  analytical  methods.  In  these 
cases  too  much  stress  cannot  be  laid  upon  the  im- 
portance of  a  systematic  inspection  of  the  supply,  and 
its  relation  to  sources  of  pollution.  Optical  evidence 
of  more  or  less  dangerous  contamination  may  often  be 
obtained  when  laboratory  methods  fail  to  detect  them. 
The  reasons  for  such  failure,  in  addition  to  those  already 
given,  are  obvious — the  polluting  matters  are  often  so 
diluted  by  the  large  mass  of  water  into  which  they  find 
their  way  as  to  be  beyond  recognition  by  the  tests 
usually  employed  in  such  work,  and  still  be  present  in 
amounts  sufficient  to  originate  disease. 

THE   QUALITATIVE    BACTERIOLOGICAL    ANALYSIS 


BACTERIOLOGICAL  STUDY  OF   WATER.       623 

OF  WATEK. — The  qualitative  bacteriological  analysis 
of  water  entails  much  labor,  as  it  requires  not  only 
that  all  the  different  species  of  organisms  found  in  the 
water  should  be  isolated,  but  that  each  representative 
should  be  subjected  to  systematic  study,  and  its  patho- 
genic or  non-pathogenic  properties  determined. 

For  this  purpose  a  knowledge  of  the  methods  for  the 
isolation  of  individual  species  which  have  already  been 
described,  and  of  the  means  of  studying  these  species 
when  isolated,  is  indispensable. 

For  this  analysis  certain  precautions  essential  to 
accuracy  are  always  to  be  observed. 

The  sample  is  to  be  collected  under  the  most  rigid 
precautions  that  will  exclude  organisms  from  sources 
other  than  that  under  consideration.  If  drawn  from  a 
spigot,  it  should  never  be  collected  until  the  water  has 
been  flowing  for  fifteen  to  twenty  minutes  in  a  full 
stream.  If  obtained  from  a  stream  or  a  spring,  it 
should  be  collected,  not  from  the  surface,  but  rather 
from  about  one  foot  beneath  the  surface. 

It  should  always  be  collected  in  vessels  which  have 
previously  been  thoroughly  freed  from  all  dirt  and 
organic  particles,  and  then  sterilized ;  and  the  plates 
should  be  made  as  quickly  as  possible  after  collecting 
the  sample. 

When  circumstances  permit,  all  water  analyses  should 
be  made  on  the  spot  where  the  sample  is  taken,  as  it 
is  known  that  during  transportation,  unless  the  samples 
are  kept  packed  in  ice,  a  multiplication  of  the  organ- 
isms contained  in  it  always  occurs. 

For  the  purpose  of  qualitative  analysis  it  is  necessary 
that  a  small  portion  of  the  water — one,  two,  three,  five 
drops — should  first  be  employed  for  making  the  plates. 


624  BACTERIOLOGY. 

In  this  way  one  can  form  an  idea  as  to  the  approximate 
number  of  organisms  in  the  water,  and  can,  in  conse- 
quence, determine  the  amount  of  wrater  best  suited  for 
the  plates.  Duplicate  plates  are  always  to  be  made — 
one  set  upon  agar-agar,  which  are  to  be  kept  in  the 
incubator  at  body-temperature,  and  one  set  upon  gelatin, 
to  be  kept  at  from  18°  to  20°  C. 

As  soon  as  colonies  have  developed  the  plates  arc  to 
be  carefully  compared  and  studied.  It  is  to  be  noted 
if  any  difference  in  the  appearance  of  the  organisms  on 
corresponding  plates  exists,  and  if  so,  to  what  it  is  due. 
It  is  to  be  particularly  noted  which  plates  contain  the 
greater  number  of  colonies,  those  kept  at  the  higher  or 
those  at  the  lower  temperature.  In  this  way  the  tem- 
perature best  suited  for  the  growth  of  the  majority  of 
these  organisms  may  be  determined.  As  a  rule,  the 
greater  number  of  colonies  appear  upon  the  gelatin 
plates  kept  at  18°  to  20°  C. ;  and  from  this  it  would 
seem  that  many  of  the  normal  water-bacteria  do  not 
find  the  higher  temperature  so  favorable  to  their  de- 
velopment as  do  the  organisms  not  naturally  present  in 
water,  particularly  the  pathogenic  varieties.  From  these 
plates  the  different  species  are  to  be  isolated  in  pure 
culture,  the  morphological  and  cultural  characteristics 
determined,  and  finally,  by  tests  upon  animals,  it  is  to 
be  decided  if  any  of  them  possess  disease-producing 
properties. 

NOTE. — What  use  should  be  made  of  this  observa- 
tion in  examining  water  for  the  presence  of  pathogenic 
bacteria  ? 

The  waters  most  frequently  studied  from  the  quali- 
tative bacteriological  standpoint  are  those  suspected 


BACTERIOLOGICAL  STUDY  OF   WATER.       625 

of  containing  specific  pathogenic  bacteria — i.  e.,  waters 
polluted  with  sewage  and  with  human  excreta  that 
are  believed  to  be  the  source  of  infection  of  typhoid 
fever,  or,  less  frequently,  of  Asiatic  cholera.  In  the 
investigations  of  such  waters  there  are  several  points  of 
which  we  should  never  lose  sight,  viz.,  unless  the 
water  is  under  continuous  study  there  is  only  a  chance 
of  detecting  the  specific  pathogenic  species,  for,  as  a  rule, 
the  dangerous  pollution  occurs  either  but  once  or  is 
intermittent,  so  that  even  in  the  case  of  exposed  streams 
there  are  periods  when  no  specifically  dangerous  con- 
tamination may  be  in  operation.  As  stated  above,  atten- 
tion is  commonly  called  to  the  water  when  the  disease, 
presumably  caused  by  its  use,  is  fully  developed,  and 
this  is  often  days  or  weeks  after  the  pollution  of  the 
stream  really  occurred.  By  an  analysis  made  at  this 
time  one  could  scarcely  hope  to  detect  the  specific  organ- 
isms that  had  caused  the  disease.  The  organisms  sought 
for  may  have  been  present  in  the  water  and  may  have 
infected  the  users,  and  yet  have  disappeared  by  the  time 
the  sample  taken  for  analysis  was  collected. 

When  present  in  polluted  waters  pathogenic  bacteria 
are  always  vastly  in  the  minority.  Were  they  con- 
stantly present  in  large  numbers  infection  among  the 
users  of  such  waters  would  be  more  frequent  and  more 
widespread  than  is  commonly  the  case.  They  may  be 
present  in  a  water-supply  in  small  numbers  ;  they  may 
even  be  in  the  sample  supplied  for  analysis,  and  yet  es- 
cape detection  if  only  the  ordinary  direct  plate  method 
of  isolation  be  used. 

From  these  considerations  it  is  obvious  that  before 
attempts  are  made  to  isolate  the  various  species  directly 
from  a  suspicious  sample  of  water  it  is  advisable  to 
40 


626  B  A  CTERIOL  OG  Y. 

subject  it  to  some  method  of  treatment  that  will  aid  in 
separating  the  few  specific  pathogenic  from  the  numer- 
ous common  saprophytic  species.  For  this  purpose 
numerous  methods  have  been  devised.  The  most  use- 
ful of  these  aim  to  favor  the  rapid  multiplication  of 
pathogenic  forms  that  may  be  present  and  to  suppress 
or  check  the  growth  of  the  ordinary  water  saprophytes. 

Attention  has  been  called  to  the  fact  that  when  ex- 
posed to  the  body-temperature  many  of  the  ordinary 
water-bacteria  develop  only  slowly  or  not  at  all,  while 
under  similar  circumstances  the  disease-producing  spe- 
cies develop  most  luxuriantly.  Advantage  has  been 
taken  of  this  observation  in  devising  methods  for  this 
particular  work,  of  which  some  of  the  following  will 
prove  serviceable : 

Collect  in  a  sterilized  flask  a  sample  of  about  100  c.c. 
of  the  water  to  be  tested,  and  add  to  this  about  25  c.c. 
of  sterilized  bouillon  of  four  times  the  usual  strength. 
This  is  then  placed  in  the  incubator  at  37°  to  38°  C., 
for  thirty-six  to  forty-eight  hours,  after  which  plates  are 
to  be  made  from  it  in  the  usual  way ;  the  results  will 
often  be  a  pure  culture  of  some  single  organism,  either 
one  of  the  intestinal  variety  or  a  closely  allied  species. 
By  a  method  analogous  to  the  latter  the  spirillum  of 
Asiatic  cholera  has  been  isolated  from  water  (see  pages 
454,  467)  ;  and  by  taking  advantage  of  the  effect  of  ele- 
vated temperature  upon  the  bacteria  of  water  Yaughan 
has  succeeded  in  isolating  from  suspicious  waters  a 
group  of  organisms  very  closely  allied  to  the  bacillus 
of  typhoid  fever. 

Theobald  Smith  has  suggested  a  method  by  which  it 
is  easily  possible  to  isolate,  from  waters  in  which  they 
are  present,  certain  organisms  that  are  of  the  utmost 


BACTERIOLOGICAL  STUDY  OF   WATER.       627 

importance  in  influencing  our  judgment  upon  the  fitness 
of  the  water  for  domestic  use.  By  the  addition  of  small 
quantities — one,  two,  or  three  drops — of  the  suspicious 
water  to  fermentation-tubes  (see  article  an  Fermenta- 
tion-tube) containing  bouillon  to  which  2  per  cent,  of 
glucose  has  been  added,  and  keeping  them  at  the  tempera- 
ture of  the  body  (37°  to  38°  C.),  the  growth  of  intestinal 
bacteria  that  may  be  present  in  the  water  is  favored, 
while  that  of  the  water-organisms  is  not ;  in  consequence, 
after  from  thirty-six  to  forty-eight  hours  the  fer- 
mentation-characteristics of  most  of  these  organisms  is 
evidenced  by  the  accumulation  of  gas  in  the  closed  end 
of  the  tube.  From  these  tubes  the  growing  bacteria 
can  then  be  easily  isolated  by  the  plate  method,  and 
intestinal  bacteria  will  not  infrequently  be  found 
present. 

For  the  isolation  of  the  typhoid  bacillus,  especially  from 
water,  a  host  of  other  methods  have  been  devised.  Some 
of  these  aim,  through  the  addition  of  special  chemical 
reagents  to  the  media,  to  retard  the  development  of 
ordinary  saprophytes  without  interrupting  the  growth 
of  the  colon  and  the  typhoid  bacillus.  Most  of  these 
methods  have  proved  disappointing.  One  of  them,  that 
of  Parietti,  still  finds  favor  in  the  hands  of  some.  It 
consists  in  adding  to  the  culture-media  to  be  used  in 
the  test  varying  amounts  of  the  following  mixture : 

Phenol 5  grammes. 

Hydrochloric  acid 4 

Distilled  water 100  c.c. 

Of  this  solution  0.1,  0.2,  and  0.3  c.c.  are  added  re- 
spectively to  each  of  three  tubes  containing  10  c.c.  of 
nutritive  bouillon.  Several  such  sets  of  tubes  are  to  be 


628  BA  CTERIOL  OGY. 

made.  To  each  are  then  added  from  1  to  3  c.c.  of  the 
water,  and  they  are  placed  in  the  incubator  at  body-tem- 
perature. It  is  said  that  whatever  development  occurs 
consists  only  of  the  typhoid  or  colon  bacillus,  or  both,  if 
they  were  present  in  the  original  sample.  They  may 
then  be  isolated  and  separated  by  the  usual  plate  method, 
or,  better  still,  through  the  application  of  the  methods 
of  v.  Drigalski  and  Conradi,  of  Ficker,  or  of  Hoffmann 
and  Ficker,  or  several  of  these  methods  in  conjunction, 
detailed  in  the  chapter  on  bacillus  typhosus.  Personally 
we  have  not  had  much  success  with  the  Parietti  method. 
The  typhoid  bacillus  has  been  isolated  from  water  by 
passing  very  large  quantities  of  water  through  an  ordi- 
nary Pasteur  or  Berkefeld  filter,  brushing  off  the  matters 
collected  on  the  filter  into  a  sterilized  vessel  and  examin- 
ing this  by  plate  methods. 

It  has  occurred  to  us  that  possibly  the  employment 
of  chemical  coagulants,  such  as  alum  and  iron,  might 
prove  serviceable  for  this  purpose.  Their  action  would 
be  to  mechanically  drag  down,  in  precipitating  as  hy- 
droxides, the  suspended  bacteria  contained  in  the  fluid. 
This  precipitate  could  then  be  examined  bacteriologi- 
cally,  instead  of  the  water,  and  the  recent  experiments 
of  Ficker  (loc.  cit.)  appear  to  demonstrate  the  value  of 
such  a  procedure. 

The  difficulties  in  this  field  of  work  are  obviously 
due  to  the  suspension  of  a  very  small  number  of  the 
disease-producing  organisms  sought  for  in  large  volumes 
of  fluid,  and  the  association  with  them  of  large  numbers 
of  other  species  that  offer  a  very  great  obstacle  to  the 
successful  search  for  the  pathogenic  varieties. 

If  by  either  of  the  above  procedures  bacilli  that  bear 
any  resemblance  to  bacillus  typhosus  be  isolated,  re- 


BACTERIOLOGICAL  STUDY  OF  WATER.       629 

course   must  then  be   had   to  all  the  differential  tests 
detailed  in  the  chapter  on  that  organism. 

THE  QUANTITATIVE  ESTIMATION  OF  BACTERIA  IN 
WATER. — Quantitative  analysis  requires  more  care  in 
the  measurement  of  the  exact  volume  of  water  em- 
ployed, for  the  results  are  to  be  expressed  in  terms  of  the 
number  of  individual  organisms  to  a  definite  volume. 
The  necessity  for  making  the  plates  at  the  place  at 
which  the  sample  is  collected  is  to  be  particularly 
accentuated  in  this  analysis,  for  multiplication  of  the 
organisms  during  transit  is  so  great  that  the  results 
of  analyses  made  after  the  water  has  been  in  a  vessel 
for  a  day  or  two  are  often  very  different  from  those  that 
would  have  been  obtained  on  the  spot. 

NOTE. — Inoculate  a  tube  containing  about  ten  cubic 
centimetres  of  sterilized  distilled  or  tap  water  with  a 
very  small  quantity  of  a  solid  culture  of  some  one  of 
the  organisms  with  which  you  have  been  working, 
taking  care  that  none  of  the  culture-medium  is  intro- 
duced into  the  water-tube  and  that  the  bacteria  are 
evenly  distributed  through  it.  Make  plates  at  once 
from  this  tube,  and  on  each  succeeding  day  determine 
by  counts  whether  there  is  an  increase  or  diminu- 
tion in  the  number  of  organisms — *.  e.,  if  they  are 
growing  or  dying.  Represent  the  results  graphically, 
and  it  will  be  noticed  that  in  many  cases  there  is 
during  the  first  three  or  four  days  a  multiplication, 
after  which  there  is  a  rapid  diminution ;  and,  if  the 
organism  does  not  form  spores,  usually  death  in  from 
ten  to  twelve  days.  This  is  not  true  for  all  organisms, 
but  does  hold  for  many. 


630 


BACTERIOLOGY. 


ENTZStSONS 


Where   it  is   not   convenient,  however,  to   make  the 
analysis    on    the    spot,  the  sample   of  water  should  be 

taken  and  -packed  in  ice  and  kept  on 
FIG.  92.  . 

ice  until  the  plates  can  be  made  from 

it,  which  should  in  all  cases  be  as  soon 
after  its  collection  as  possible. 

For  the  collection  of  samples  from 
the  deeper  portions  of  streams,  lakes, 
etc.,  a  number  of  convenient  devices 
have  been  made.  A  very  satisfactory 
apparatus  has  been  made  for  me  by 
Messrs.  Charles  Lcntz<&  Sons,  of  Phila- 
delphia. It  consists  of  a  metal  frame- 
work, in  which  is  encased  a  bottle 
provided  with  a  ground-glass  stopper. 
To  the  stopper  a  spring  clamp  is  at- 
tached, and  this  in  turn  is  operated  by 
a  string,  so  that  when  the  weighted 
apparatus  is  allowed  to  sink  into  the 
stream  the  stopper  may  be  removed 
from  the  bottle  at  any  depth  by  simply 
pulling  upon  the  .string.  When  the 
bottle  is  filled  with  water  the  stopper 
is  allowed  to  spring  back  into  position 
by  releasing  the  string.  The  whole 
apparatus  (depicted  in  Fig.  92)  is  pro- 
vided with  a  weight  that  insures  its 
sinking,  and  a  heavy  cord  by  which  it  may  be  lowered 
and  raised.  It  should  be  sterilized  before  using.  After 
collecting  the  sample  the  bottle  should  be  wiped  dry 
with  a  sterilized  towel.  Before  removing  the  stopper 
the  mouth  of  the  bottle  should  be  rinsed  with  alcohol 


Bottle  for  collecting 
water. 


BACTERIOLOGICAL  STUDY  OF   WATER.       631 

and  heated  with  a  gas-flame,  to  prevent  contamination 
of  its  contents  by  matters  that  may  have  been  upon 
its  surface. 

In  beginning  the  quantitative  analysis  of  water  with 
which  one  is  not  acquainted  certain  preliminary  steps 
are  essential. 

It  is  necessary  to  know  approximately  the  number  of 
organisms  contained  in  any  fixed  volume,  so  as  to  deter- 
mine the  quantity  of  water  to  be  employed  for  the  plates 
or  tubes.  This  is  usually  done  by  making  preliminary 
plates  from  one  drop,  two  drops,  0.25  c.c.,  0.5  c.c.,  and 
1  c.c.  of  the  water.  After  each  plate  has  been  labelled 
with  the  amount  of  water  used  in  making  it,  it  is  placed 
aside  for  development.  When  this  has  occurred  one 
selects  the  plate  upon  which  the  colonies  are  only  mod- 
erate in  number — about  200  to  300  colonies  presenting 
— and  employs  in  the  subsequent  analysis  the  same 
amount  of  water  that  was  used  in  making  this  plate. 

If  the  original  water  contained  so  many  organisms 
that  there  developed  on  a  plate  or  tube  made  with  one 
drop  too  many  colonies  to  be  easily  counted,  then  the 
sample  must  be  diluted  with  one,  ten,  twenty-five,  fifty, 
or  one  hundred  volumes,  as  the  case  may  require,  of 
sterilized  distilled  water.  This  dilution  must  be  accu- 
rate, and  its  exact  extent  noted,  so  that  subsequently  the 
number  of  organisms  per  volume  in  the  original  water 
may  be  calculated. 

The  use  of  a  drop  is  not  sufficiently  accurate.  The 
dilution  should  therefore  always  be  to  a  degree  that  will 
admit  of  the  employment  of  a  volume  of  water  that 
may  be  exactly  measured,  0.25  and  0.5  c.c.  being  the 
amounts  most  convenient  for  use. 

Duplicate  plates  should   always   be    made,  and  the 


632  B  A  CTERIOL  OGY. 

mean  of  the  number  of  colonies  that  develop  upon 
them  taken  as  the  basis  from  which  to  calculate  the 
number  of  organisms  per  volume  in  the  original  water. 

For  example :  from  a  sample  of  water  0.25  c.c.  is 
added  to  a  tube  of  liquefied  gelatin,  carefully  mixed  and 
poured  as  a  plate.  When  development  occurs  the 
number  of  colonies  is  too  numerous  to  be  accurately 
counted.  One  cubic  centimetre  of  the  original  water 
is  then  to  have  added  to  it,  under  precautions  that  pre- 
vent contamination  from  without,  99  c.c.  of  sterilized 
distilled  water — that  is,  we  have  now  a  dilution  of 
1  :  100.  Again,  0.25  c.c.  of  this  dilution  is  plated, 
and  we  find  180  colonies  on  the  plate.  Assuming  that 
each  colony  develops  from  an  individual  bacterium, 
though  this  is  perhaps  not  strictly  true,  we  had  180 
organisms  in  0.25  c.c.  of  our  1  :  100  dilution ;  therefore 
in  0.25  c.c.  of  the  original  water  we  had  180  X  100  = 
18,000  bacteria,  which  will  be  72,000  bacteria  per  cubic 
centimetre  (0.25  c.c.  =  18,000,  1  c.c.  =  18,000  X  4  = 
72,000).  The  results  are  always  to  be  expressed  in 
terms  of  the  number  of  bacteria  per  cubic  centimetre 
of  the  original  water. 

Another  point  of  very  great  importance  (already  men- 
tioned) is  the  effect  of  temperature  upon  the  number  of 
colonies  of  bacteria  that  will  develop  on  the  plates  made 
from  water.  It  must  always  be  remembered  that  a 
larger  number  of  colonies  appear  on  gelatin  plates  made 
from  water  and  kept  at  18°  to  20°  C.  than  on  agar-agar 
plates  kept  in  the  incubator.  The  following  table,  illus- 
trative of  this  point,  gives  the  results  of  parallel  anal- 
yses of  the  same  waters,  the  one  series  of  counts  having 
been  made  upon  gelatin  plates  at  the  ordinary  tempera- 
ture of  the  room,  the  other  upon  plates  of  agar-agar 


BACTERIOLOGICAL  STUDY  OF   WATER.       633 

kept  for  the  same  length  of  time  in  the  incubator  at 
from  37°  to  38°  C.  It  will  be  seen  from  the  table 
that  much  the  larger  number  of  colonies — i.  e.,  much 
higher  results — were  always  obtained  when  gelatin  was 
employed.  The  importance  of  this  point  in  the  quan- 
titative bacteriological  analysis  of  water  is  too  apparent 
to  require  further  comment. 

TABLE  COMPARING  THE  RESULTS  OBTAINED  BY  THE  USE  OF  GEL- 
ATIN AT  18°-20°  C.  AND  AGAR-AGAR  AT  37°-38°  C.  IN  QUANTI- 
TATIVE BACTERIOLOGICAL  ANALYSES  OF  WATER.-  RESULTS 
RECORDED  ARE  THE  NUMBER  OF  COLONIES  THAT  DEVELOPED 
FROM  THE  SAME  AMOUNT  OF  VARIOUS  WATERS  IN  EACH 
SERIES.* 

NUMBER  OF  COLONIES  FROM  WATER  THAT  DEVELOPED  UPON— 
oeiatin  plates  at  18°  to  20°  C.  Agar-agar  plates  at  37°  to  38°  C. 

.310  170 

280  140 

310) f  180 

340) U60 

650) f210 

630) (320 

380) J290 

400  j -  1 210 

1000) (100 

890) J130 

340) r  280 

370) (210 

490  I ...    ...f  110 

580 1 (100 

Another  point  of  equal  importance  in  its  influence  upon 
the  number  of  colonies  that  develop  is  the  reaction  of  the 
gelatin.  A  marked  excess  of  either  alkalinity  or  acidity 
always  has  a  retarding  effect  upon  many  species  found  in 
water.  Fuller's  experience  at  the  Lawrence  (Mass.)  Ex- 

1  I  am  indebted  to  James  Homer  Wright,  Thomas  Scott  Fellow  in 
Hygiene  (1892-'93),  University  of  Pennsylvania,  for  the  results  pre- 
sented in  this  table. 


634  £A  CTER 10  LOGY. 

perirnent  Station  lias  shown  that  gelatin  of  such  a  degree 
of  acidity  as  to  require  t\\v  further  addition  of  from  15  to 
20  c.c.  per  litre  of  a  normal  caustic  alkali  solution  to  bring 
it  to  the  phenolphtalein  neutral  point  gives,  on  the  whole, 
the  best  results.  Thus,  by  way  of  illustration,  Fuller 
found  that  a  sample  of  Merrimac  River  water  gave 
5800  colonies  per  c.c.  on  phenolphtalein  neutral  gel- 
atin, 15,000  colonies  on  gelatin  that  would  need  20  c.c. 
of  normal  alkali  solution  to  bring  it  up  to  the  phenol- 
phtalein neutral  point — i.  e.,  a  feebly  acid  nutrient  gel- 
atin, and  500  colonies  on  a  gelatin  so  alkaline  as  to 
require  20  c.c.  of  a  normal  acid  solution  to  bring  it 
back  to  the  phenolphtalein  neutral  point. 

Throughout  this  part  of  the  work  it  is  to  be  borne  in 
mind  that  when  reference  is  made  to  plates  it  is  not  to 
a  set,  as  in  isolation  experiments,  but  to  a  single  plate. 

METHOD  OF  COUNTING  THE  COLONIES  ON  PLATES. 
— For  convenience  in  counting  colonies  on  plates  or  in 
tubes  it  is  customary  to  divide  the  whole  area  of  the 
gelatin  occupied  by  colonies  into  smaller  areas,  and 
either  count  all  the  colonies  in  each  of  these  areas  and 
add  the  several  sums  together  for  the  total,  or  to  count 
the  number  of  colonies  in  each  of  several  areas,  ten 
or  twelve,  take  the  mean  of  the  results  and  multiply 
this  by  the  number  of  areas  containing  colonies.  The 
latter  procedure  obtains,  of  course,  only  when  all  the 
areas  are  of  the  same  size.  By  this  method,  however, 
the  results  vary  so  much  in  different  counts  of  the  same 
plate  that  they  cannot  be  considered  as  more  than  rough 
approximations. 

NOTE. — Prepare  a  plate ;  calculate  the  number  of 
colonies  upon  it  by  this  latter  method.  Now  repeat 


BACTERIOLOGICAL  STUDY  OF  WATEU.       635 

the  calculation,  making  the  average  from  another  set 
of  squares.  Now  actually  count  the  entire  number  of 
colonies  on  the  plate.  Compare  the  results. 

For  facilitating  the  counting  of  colonies  several  very 
convenient  devices  exist. 

WOLFFHUGEL'S  COUNTING-APPARATUS. — This  appa- 
ratus (Fig.  93)  consists  of  a  flat  wooden  stand,  the 
centre  of  which  is  cut  out  in  such  a  way  that  either  a 
black  or  white  glass  plate  may  be  placed  in  it.  These 
form  a  background  upon  which  the  colonies  may  more 
easily  be  seen  when  the  plate  to  be  counted  is  placed 


FIG.  93. 


Wolff  hugel's  apparatus  for  counting  colonies. 

upon  it.  When  the  gelatin  plate  containing  the  colonies 
has  been  placed  upon  this  background  of  glass  it  is 
covered  by  a  transparent  glass  plate  which  swings  on  a 
hinge.  This  plate,  which  is  ruled  in  square  centimetres 
and  subdivisions,  when  in  position  is  just  above  the 
colonies,  without  touching  them.  The  gelatin  plate  is 
moved  about  until  it  rests  under  the  centre  of  the  area 
occupied  by  the  ruled  lines.  The  number  of  colonies 


636  EA  CTERIOLOG  Y. 

in  each  square  centimetre  is  then  counted,  and  the  sum- 
total  of  the  colonies  in  all  these  areas  gives  the  number 
of  colonies  on  the  plate ;  or,  as  has  already  been  indi- 
cated, if  the  number  of  colonies  be  very  great,  a  mean 
may  be  taken  of  the  number  in  several  (six  or  eight) 
squares ;  this  is  to  be  multiplied  by  the  total  number 
of  squares  occupied  by  the  gelatin.  The  result  is  an 
approximation  of  the  total  number  of  colonies. 

When  the  colonies  are  quite  small,  as  is  frequently 
the  case,  the  counting  may  be  rendered  easier  by  the 
use  of  a  small  hand  lens.  (Fig.  94.) 

FIG.  94. 


Lens  for  counting  colonies. 

Several  useful  modifications  of  the  apparatus  of  Wolff- 
hugel  have  been  introduced.  The  most  important  is 
that  of  Lafar.1  Lafar's  counter  consists  of  a  glass  disk 
of  the  diameter  of  ordinary  size  Petri  dishes.  It  is 
supplied  with  a  collar  or  flange  that  fits  around  the 
bottom  of  the  Petri  dish,  and  thus  holds  the  counter  in 
position.  The  disk  is  ruled  with  concentric  circles,  and 
its  area  is  divided  into  sectors  of  such  sizes  that  the 
spaces  between  the  concentric  circles  and  the  radii  form- 
ing the  sectors  are  of  equal  size.  Three  of  the  sectors 
are  subdivided  into  smaller  areas  of  equal  size  for  con- 
venience in  counting  when  the  colonies  are  very  numer- 
ous. The  principles  involved  are  similar  to  those  of  the 

1  Lafar:  Ceutralblatt  fur  Bakteriologie  und  Parasitenknude,  1891, 
Bd.  xv.  S.  331. 


BACTERIOLOGICAL  STUDY  OF   WATER.       637 

preceding  apparatus,  but  the  circular  form  of  the  appa- 
ratus admits  of  more  exactness  when  counting  colonies 
on  a  circular  plate.1 

Pakes 2  has  introduced  a  cheap  and  convenient  modi- 
fication of  Lafar's  apparatus.  It  consists  of  a  sheet  of 
white  paper  on  which  is  printed  a  black  disk  ruled 


-  

8 

Pakes's  apparatus  for  counting  colonies  (reduced  one-third). 

with  white  lines,  in  somewhat  the  same  fashion  as  is 
Lafar's  counter,  though  the  areas  of  the  smallest  sub- 
divisions are  not  of  one  size  and  do  not  bear  a  constant 

1  Lafar's  apparatus  is  to  be  obtained  from  F.  Mollenkopf,  10  Thor- 
strasse,   Stuttgart,  who  holds  the  patent  for  it.     Its  price  is  about  8 
marks. 

2  Journal  of  Bacteriology  and  Pathology,  1896,  vol.  iv.  No.  1. 


638  B  A  CTER IOL  OGY. 

relation  to  each  other.1  To  use  this  apparatus  (Fig. 
95)  the  Petri  dish  is  placed  centrally  upon  it,  the 
cover  of  the  dish  is  removed,  and  the  colonies  are 
counted  as  they  lie  over  the  spaces  bounded  by  the 
white  lines  on  the  black  disk  beneath.  When  the 
plate  is  centered  over  the  black  disk  the  portion  lying 
over  one  sector  is  exactly  one-sixteenth  of  the  whole 
plate. 

ESMARCH'S  COUNTER. — Esmarch  devised  a  counter 
(Fig.  96)  for  estimating  the  number  of  colonies  present 

FIG.  96. 


Esmarch's  apparatus  for  counting  colonies  in  rolled  tubes. 

upon  a  cylindrical  surface,  as  when   in   rolled  tubes. 
The  principles  and  methods  of  estimation  are  practically 
the  same  as  those  given  for  WolfFhiigePs  apparatus. 
A  simpler  method  than   by  the  use  of  Esmarch's 

1  Copies  of  this  apparatus  are  to  be  had  of  Ash  &  Co.,  42  Southwark 
Street,  London,  or  of  Lentz  &  Sons,  North  Eleventh  Street,  Phila- 
delphia, Pa.  (The  cost  is  but  a  few  cents  per  copy.) 


BACTERIOLOGICAL  STUDY  OF   WATER.       639 

apparatus  may  be  employed  for  counting  the  colonies 
in  rolled  tubes.  It  consists  in  dividing  the  tube  by 
lines  into  four  or  six  longitudinal  areas,  which  are  sub- 
divided by  transverse  lines  about  1  or  2  cm.  apart.  The 
lines  may  be  drawn  with  pen  and  ink.  They  need  not 
be  exactly  the  same  distance  apart  nor  exactly  straight. 
Beginning  with  one  of  these  squares  at  one  end  of  the 
tube,  which  may  be  marked  with  a  cross,  the  tube  is 
twisted  with  the  fingers,  always  in  one  direction,  and 
the  exact  number  of  colonies  in .  each  square  as  it 
appears  in  rotation  is  counted,  care  being  taken  not  to 
count  a  square  more  than  once  ;  the  sums  are  then  added 
together,  and  the  result  gives  the  number  of  colonies  in 
the  tube.  This  method  may  be  facilitated  by  the  use 
of  a  hand-lens. 

In  all  these  methods  there  is  one  error  difficult  to 
eliminate  :  it  is  assumed  that  each  colony  has  grown 
from  a  single  organism.  This  is  probably  not  always 
the  case,  as  there  may  exist  clumps  of  bacteria  which 
represent  hundreds  or  even  thousands  of  individuals, 
but  which  still  give  rise  to  but  a  single  colony — ob- 
viously this  is  of  necessity  estimated  as  a  single  organ- 
ism in  the  water  under  analysis. 

Where  grounds  exist  for  suspecting  the  presence  of 
these  clumps  they  may  in  part  be  broken  up  by  shaking 
the  original  water  with  sterilized  sand. 

What  has  been  said  for  the  bacteriological  examina- 
tion of  water  holds  good  for  all  fluids  which  are  to  be 
subjected  to  this  form  of  analysis. 

THE  SEWAGE  STREPTOCOCCUS. — Houston1  reached 
the  conclusion  that  there  is  constantly  present  in  sewage 
a  particular  form  of  streptococcus  which  is  really  more 

1  Houston  :  Ann.  Report,  Local  Gov.  Board,  xxviii. 


640  BACTERIOLOGY. 

positively  indicative  of  the  contamination  of  water 
by  sewage  than  is  bacillus  coli.  This  opinion  has 
recently  been  under  investigation  by  members  of  the 
staff  of  the  Massachusetts  Institute  of  Technology,  and 
they  have  reached  the  conclusion  that  considerable 
reliance  can  be  placed  upon  the  presence  of  this  organ- 
ism as  an  indication  of  sewage  pollution  of  water. 

The  presence  of  the  sewage  streptococcus  is  most 
readily  shown  in  the  sediment  in  fermentation  tubes 
inoculated  with  water  under  examination.  If  the  sew- 
age streptococcus  is  present  it  is  very  easy  to  demon- 
strate it  by  microscopic  examination  of  the  sediment  after 
twenty-four  to  forty-eight  hours.  In  addition  to  this 
test  it  has  also  been  demonstrated  by  Winslow l  that  the 
estimation  of  the  degree  of  acidity  of  the  contents  of  the 
fermentation  tube  is  a  safe  indication  of  the  presence  of 
the  sewage  streptococcus.  When  this  organism  is  pres- 
ent the  acidity  rises  far  more  rapidly  and  to  a  greater 
height  than  is  the  case  when  it  is  absent,  so  that  in  this 
way  an  additional  indicator  is  available  as  to  the  pota- 
bility of  a  water  under  examination. 

BACTERIOLOGICAL   ANALYSIS   OF   AIR. 

Quite  a  number  of  methods  for  the  .bacteriological 
study  of  the  air  exist.  In  the  main  they  consist  either 
in  allowing  air  to  pass  over  solid  nutrient  media  (Koch, 
Hesse)  and  observing  the  colonies  which  develop  upon 
the  media,  or  in  filtering  the  bacteria  from  the  air  by 
means  of  porous  and  liquid  substances,  and  studying  the 
organisms  thus  obtained.  (Miguel,  Petri,  Strauss,  Wiirz, 
Sedgwick-Tucker.)  Because  of  their  greater  exactness, 
the  latter  have  supplanted  the  former  methods. 

1  Winslow :  Jour.  Mecl.  Research,  vol.  iii.,  1902. 


BACTERIOLOGICAL  ANALYSIS  OF  AIR.        641 

In  some  of  the  methods  which  provide  for  the  filtra- 
tion of  bacteria  from  the  air  by  means  of  liquid  sub- 
stances a  measured  volume  of  air  is  aspirated  through 
liquefied  gelatin ;  this  is  then  rolled  into  an  Esmarch 
tube  and  the  number  of  colonies  counted,  just  as  is 
done  in  water  analysis.  This  is  the  simplest  procedure. 
An  objection  sometimes  raised  against  it  is  that  organisms 

FIG.  97. 


Petri's  apparatus  for  bacteriological  analysis  of  air.    The  tube 
packed  with  sand  is  seen  at  the  point  a. 

may  be  lost,  and  not  come  into  the  calculation,  by  pass- 
ing through  the  medium  in  the  centre  of  an  air-bubble 
without  being  arrested  by  the  fluid — an  objection  that 
appears  to  have  more  of  speculative  than  of  real  value. 
Filtration  through  porous  substances  appears,  on  the 
whole,  to  give  the  best  results.  Petri  recommends  as- 
piration of  a  measured  volume  of  air  through  glass 
tubes  into  which  sterilized  sand  is  packed.  (Fig.  97.) 
When  aspiration  is  finished  the  sand  is  mixed  with 
liquefied  gelatin,  plates  are  made,  and  the  number  of 
developing  colonies  counted,  the  results  giving  the 
41 


642  BACTERIOLOGY. 

number  of  organisms  contained  in  the  volume  of  air 
aspirated  through  the  sand. 

The  main  objection  to  this  method  is  the  possibility 
of  mistaking  a  sand-granule  for  a  colony.  This  objec- 
tion has  been  overcome  by  Sedgwick  and  Tucker,  who 
employ  granulated  sugar  instead  of  sand ;  this,  when 
brought  into  the  liquefied  gelatin,  dissolves,  and  no  such 
error  as  that  possible  in  the  Petri  method  can  be  made. 

SEDGWICK-TUCKER  METHOD. — On  the  whole,  the 
method  proposed  by  Sedgwick  and  Tucker  gives  such 
uniform  results  that  it  is  to  be  preferred  to  others.  It 
is  as  follows  : 

The  apparatus  employed  by  them  consists  essentially 
of  three  parts : 

1.  A  glass  tube  of  special  form,  to  which  the  name 
aerobioscope  has  been  given. 

2.  A  stout  copper  cylinder  of   about  sixteen  litres 
capacity,  provided  with  a  vacuum-gauge. 

3.  An  air-pump. 

The  aerobioscope  (Fig.  98)  is  about  35  cm.  in  its 
entire  length ;  it  is  15  cm.  long  and  4.5  cm.  in  diam- 
eter at  its  expanded  part ;  one  end  of  the  expanded  part 
is  narrowed  to  a  neck  2.5  cm.  in  diameter  and  2.5  cm. 
long.  To  the  other  end  is  fused  a  glass  tube  15  cm. 
long  and  0.5  cm.  inside  diameter,  in  which  is  to  be 
placed  the  filtering-material. 

Upon  this  narrow  tube,  5  cm.  from  the  lower  end,  a 
mark  is  made  with  a  file,  and  up  to  this  mark  a  small 
roll  of  brass-wire  gauze  (a)  is  inserted ;  this  serves  as 
a  stop  for  the  filtering-material  which  is  to  be  placed 
over  it.  Beneath  the  gauze  (at  6),  and  also  at  the  J , 
large  end  (c),  the  apparatus  is  plugged  with  cotton. 
When  thoroughly  cleaned,  dried,  and  plugged,  the 


BACTERIOLOGICAL  ANALYSIS  OF  AIR.       643 

apparatus  is  to  be  sterilized  in  the  hot-air  sterilizer. 
When  cool  the  cotton  plug  is  removed  from  the  large 
end  (c),  and  thoroughly  dried  and  sterilized  No.  50 
granulated  sugar  is  poured  in  until  it  just  fills  the 
10  cm.  (d)  of  the  narrow  tube  above  the  wire  gauze. 
This  column  of  sugar  is  the  filtering-material  em- 
ployed to  engage  and  retain  the  bacteria.  After 
pouring  in  the  sugar  the  cotton- wool  plug  is  replaced, 
and  the  tube  is  again  sterilized  at  120°  C.  for  several 
hours. 

Taking   the  air   sample.     In   order   to  measure   the 
amount  of  air  used  the  value  of  each  degree   on   the 

FIG.  98. 


i  ;  ; 

d  a  o 

The  Sedgwick-Tucker  aerobioscope. 


vacuum-gauge  is  determined  in  terms  of  air  by  means 
of  an  air-meter,  or  by  calculation  from  the  known  ca- 
pacity of  the  cylinder.  This  fact  ascertained,  the  nega- 
tive pressure  indicated  by  the  needle  on  exhausting  the 
cylinder  shows  the  volume  of  air  which  must  pass  into 
it  in  order  to  fill  the  vacuum.  By  means  of  the  air- 
pump  one  exhausts  the  cylinder  until  the  needle 
reaches  the  mark  corresponding  to  the  amount  of  air 
required.1 

1  Such  a  cylinder  and  air-pump  are  not  necessary.  A  pair  of  ordinary 
aspirating-bottles  of  known  capacity  graduated  into  litres  and  fractions 
thereof  answer  perfectly  well.  Or  one  can  determine  by  the  weight 
of  water  that  has  flowed  from  the  aspirator  the  volume  of  air  that  has 
passed  in  to  take  its  place — i.  e.,  the  volume  of  air  that  has  passed 
through  the  aerobioscope. 


644  BACTERIOLOGY. 

A  sterilized  aerobioscope  is  now  to  be  fixed  in  the 
upright  position  and  its  small  end  connected  by  a  rubber 
tube  with  a  stopcock  on  the  cylinder,  or  to  a  glass  tube 
tightly  fixed  in  the  neck  of  an  aspirating-bottle  by 
means  of  a  perforated  rubber  stopper.  The  cotton  plug 
is  then  moved  from  the  upper  end  of  the  aerobioscope, 
and  the  desired  amount  of  air  is  aspirated  through  the 
sugar.  Dust-particles  and  bacteria  will  be  held  back 
by  the  sugar.  During  manipulation  the  cotton  plug  is 
to  be  protected  from  contamination. 

When  the  required  amount  of  air  has  been  aspirated 
through  the  sugar  the  cotton  plug  is  replaced,  and  by 
gently  tapping  the  aerobioscope  while  held  in  an  almost 
horizontal  position  the  sugar,  and  with  it,  the  bacteria, 
are  brought  into  the  large  part  (e)  of  the  apparatus. 
WJben  all  the  sugar  is  thus  shaken  down  into  this  part 
of  the  apparatus  about  20  c.c.  of  liquefied,  sterilized 
gelatin  is  poured  in  through  the  opening  at  the  end  c, 
the  sugar  dissolves,  and  the  whole  is  then  rolled  on  ice, 
just  as  is  done  in  the  preparation  of  an  ordinary  Esmarch 
tube. 

The  gelatin  is  most  easily  poured  into  the  aerobio- 
scope by  the  use  of  a  small,  sterilized,  cylindrical  funnel 
(Fig.  99),  the  stem  of  which  is  bent  to  an  angle  of  about 
110°  with  the  long  axis  of  the  body. 

The  larger  part  of  the  aerobioscope  is  divided  into 
squares  to  facilitate  the  counting  of  the  colonies. 

By  the  employment  of  this  apparatus  one  can  filter 
the  air  at  any  place,  and  can  then,  without  fear  of  con- 
tamination, carry  the  tubes  to  the  laboratory  and  com- 
plete the  analysis.  Aside  from  this  advantage,  the  filter 
being  soluble  only  the  insoluble  bacteria  are  left  im- 
bedded in  the  gelatin. 


BACTERIOLOGICAL  ANALYSIS  OF  AIR.        645 

For  general  use  this  method  is  to  be  preferred  to  the 
others  that  have  been  mentioned. 

BACTERIOLOGICAL    STUDY   OF   THE   SOIL. 

Bacteriological  study  of  the  soil  may  be  made  by 
either  breaking  up  small  particles  of  earth  in  liquefied 
media  and  making  plates  directly  from  this ;  or  by  what 
is  perhaps  a  better  method,  as  it  gets  rid  of  insoluble 
particles  which  may  give  rise  to  errors :  breaking  up 

FIG.  99. 


Bent  funnel  for  use  with  aerobioscope. 

the    soil   in   sterilized   water  and   then    making   plates 
immediately  from  the  water. 

It  must  be  borne  in  mind  that  many  of  the  ground- 
organisms  belong  to  the  anaerobic  group,  so  that  in 
these  studies  this  point  should  be  remembered  and  the 
methods  for  the  cultivation  of  such  organisms  practised 
in  connection  with  the  ordinary  methods.  It  must  also 
be  remembered  that  the  nitrifying  organisms,  every- 


646  BA  CTERJO  LOGY. 

where  present  in  the  ground,  cannot  be  isolated  by  the 
ordinary  methods,  and  will  not  appear  in  plates  made 
after  either  of  the  above  plans.  The  special  devices  for 
their  cultivation  are  described  in  the  chapter  on  Soil- 
organisms. 

BACTERIOLOGICAL    STUDY    OF    MILK. 

The  possibility  of  milk  serving  as  a  vehicle  in  which 
disease-producing  bacteria  may  be  disseminated  through- 
out a  community  has  long  been  recognized,  and  epi- 
demics of  typhoid  fever  have  been  traced  directly  to 
infected  milk,  while  such  diseases  as  diphtheria  and 
scarlet  fever  are  also  frequently  regarded  as  being  con- 
veyed in  the  same  manner. 

In  recent  years  the  detailed  study  of  the  milk  of 
individual  cows  has  revealed  the  fact  that  streptococcus 
mastitis  is  not  an  uncommon  occurrence  in  herds,  and 
it  has  frequently  been  observed  that  milk  rich  in  strep- 
tococci may  prove  dangerous  when  fed  to  infants  and 
convalescents. 

Since  milk  is  such  a  favorable  medium  for  the  growth 
of  a  variety  of  bacteria  it  is  not  at  all  uncommon  to 
find  market  milk  very  rich  in  bacteria,  especially  if  it 
has  been  collected  in  a  careless  manner  in  dirty  recep- 
tacles, in  unsanitary  stables,  and  has  been  shipped  long 
distances  at  comparatively  high  temperatures. 

For  these  various  reasons  the  bacteriological  study 
of  milk  has  gained  considerable  prominence  during  the 
past  few  years — so  much  so  that  in  some  localities  an 
effort  is  being  made  to  establish  a  bacterial  standard  for 
market  milk — that  is,  milk  containing  more  than  a  cer- 
tain number  of  bacteria  is  not  regarded  as  suitable  for 
use.  Whether  such  a  standard  can  be  maintained  or 


BACTERIOLOGICAL  STUDY  OF  MILK.         647 

not  remains  to  be  demonstrated.  The  several  milk 
commissions  composed  of  pediatrists  in  various  large 
cities  have  established  a  bacterial  standard  for  pediatric 
milk  of  10,000  bacteria  to  the  cubic  centimetre.  Expe- 
rience has  shown  that  it  is  possible  to  market  milk  that 
meets  this  bacterial  standard  sometimes  with  merely 
ordinary  precautions  with  regard  to  cleanliness.  In 
larger  dairies  it  has  frequently  been  a  question  of  some 
difficulty  on  account  of  the  elaborate  scale  on  which 
everything  is  conducted. 

QUANTITATIVE  BACTERIOLOGICAL  ANALYSIS. — In 
the  quantitative  bacteriological  examination  of  market 
milk  it  is  necessary  to  dilute  the  milk  with  sterile  water 
or  sterile  salt  solution  before  plating  on  account  of  the 
very  large  numbers  of  bacteria  present.  The  degree 
of  dilution  that  is  necessary  will  depend  upon  the 
nature  of  the  dairy  from  which  the  milk  is  derived,  the 
age  of  the  milk,  and  the  temperature  at  which  it  has 
been  kept.  Usually  a  dilution  of  1  to  100,  1  to  1000, 
and  1  to  10,000  is  sufficient.  From  these  dilutions 
plate  cultures  are  made  with  0.1,  0.2,  0.3  cubic  centi- 
metre of  each  dilution. 

QUALITATIVE  BACTERIOLOGICAL  ANALYSIS.  — 
Aside  from  the  quantitative  bacteriological  analysis  of 
milk  the  qualitative  analysis  has  received  a  great  deal 
of  attention.  Detailed  qualitative  analysis  necessarily 
entails  an  enormous  amount  of  labor,  but  the  detection 
of  certain  forms  of  bacteria  is  not  always  very  difficult. 
This  applies  especially  to  the  detection  of  streptococci. 

Since  milk  containing  streptococci  in  considerable 
numbers  is  derived  from  the  udder  of  a  cow  suffering 
from  some  form  of  mastitis,  it  is  always  possible  to  find 
pus  in  such  milk.  Consequently  it  is  customary  to 


648  £A  CTERIOLOG  Y. 

examine  such  milk  for  the  presence  of  both  streptococci 
and  pus.  Tins  is  done  by  centrifuging  a  cubic  centi- 
metre of  the  milk  and  collecting  the  sediment  on  a 
clean  cover-slip  and  staining  with  Loffler's  methylene- 
blue.  In  this  manner  practically  all  the  sediment  derived 
from  one  cubic  centimetre  can  be  obtained  on  the  cover- 
slip  and  a  fairly  satisfactory  estimate  can  be  made  of 
the  relative  number  of  pus  cells  in  this  quantity  of 
milk  as  well  as  at  the  same  time  an  estimation  of  the 
relative  number  of  streptococci. 

Milk  that  shows  pus  cells  along  with  distinct  chains 
of  streptococci,  either  extra-  or  intracellular,  is  usually 
regarded  as  dangerous  in  character,  and  boards  of  health 
usually  direct  that  the  cows  from  which  such  milk  is 
derived  be  excluded  from  the  dairy  until  such  time  as 
the  milk  is  free  from  these  elements. 


CHAPTER    XXVII. 

Various  experiments  in  sterilization  by  steam  and  by  hot  air. 

PLACE  in  one  of  the  openings  in  the  cover  of  the 
steam  sterilizer  an  accurate  thermometer;  when  the 
steam  has  been  streaming  for  a  minute  or  two  the  ther- 
mometer will  register  100°  C.  Wrap  in  a  bundle  of 
towels  or  rags  or  pack  tightly  in  cotton  a  maximum 
(self-registering)  thermometer ;  let  this  thermometer  be 
in  the  centre  of  a  bundle  large  enough  to  quite  fill  the 
chamber  of  the  sterilizer.  At  the  end  of  a  few  minutes7 
exposure  to  the  streaming  steam  remove  it ;  it  will  be 
found  to  indicate  a  temperature  of  100°  C. 

Closer  study  of  the  penetration  of  steam  has  taught 
us,  however,  that  the  temperature  found  at  the  centre 
of  such  a  mass  may  sometimes  be  that  of  the  air  in  the 
meshes  of  the  material,  and  not  that  of  steam,  and  for 
this  reason  the  sterilization  at  that  point  may  not  be 
complete,  because  hot  air  at  100°  C.  has  not  the  ster- 
ilizing properties  that  steam  has  at  the  same  temperature. 
It  is  necessary,  therefore,  that  this  air  should  be  ex- 
pelled from  the  meshes  of  the  material  and  its  place 
taken  by  the  steam  before  sterilization  is  complete.  This 
is  insured  by  allowing  the  steam  to  stream  through  the 
substances  a  few  minutes  before  beginning  to  calculate 
the  time  of  exposure.  There  is  as  yet  no  absolutely 
sure  means  of  saying  that  the  temperature  at  the  centre 
of  the  mass  is  that  of  hot  air  or  of  steam,  so  that  the 
exact  length  of  time  that  is  required  for  the  expulsion 


650  RA  CTERIOLOG  Y. 

of  the  air  from   the  meshes  of  the   material  cannot  be 
given. 

Determine  if  the  maximum  thermometer  indicates  a 
temperature  of  100°  C.  at  the  centre  of  a  moist  bundle 
in  the  same  way  as  when  a  dry  bundle  was  employed. 

To  about  50  c.c.  of  bouillon  add  about  1  gramme 
of  chopped  hay,  and  allow  it  to  stand  in  a  warm  place 
for  twenty-four  hours.  At  the  end  of  this  time  it  will 
be  found  to  contain  a  great  variety  of  organisms.  Con- 
tinue the  observation,  and  ultimately  a  pellicle  will  be 
seen  to  form  on  the  surface  of  the  fluid.  This  pellicle 
is  made  up  of  rods  which  grow  as  long  threads  in 
parallel  strands.  In  many  of  these  rods  glistening 
spores  will  be  seen.  After  thoroughly  shaking,  filter 
the  mass  through  a  fine  cloth  to  remove  coarser  parti- 
cles. 

Pour  into  each  of  several  test-tubes  about  10  c.c.  of 
the  filtrate.  Allow  one  tube  to  remain  undisturbed  in  a 
warm  place.  Place  another  in  the  steam  sterilizer  for 
five  minutes  ;  a  third  for  ten  minutes  ;  a  fourth  for  one- 
half  hour ;  a  fifth  for  one  hour. 

At  the  end  of  each  of  these  exposures  inoculate  a 
tube  of  sterilized  bouillon  from  each  tube.  Likewise 
make  a  set  of  plates  or  Esmarch  tubes  upon  both  gel- 
atin and  agar-agar  from  each  tube,  and  note  the  results. 
At  the  same  time  prepare  a  set  of  plates  or  Esmarch 
tubes  on  agar-agar  and  on  gelatin  from  the  tube  which 
has  not  been  exposed  to  the  action  of  the  steam. 

The  plates  or  tubes  from  the  unmolested  tube  will 
present  colonies  of  a  variety  of  organisms  ;  separate  and 
study  these. 

Those  from  the  tube  which  has  been  sterilized  for 


STERILIZATION  BY  STEAM  AND  HOT  AIR.   651 

five  minutes  will  present  colonies  in  moderate  numbers ; 
but,  as  a  rule,  they  will  represent  but  a  single  organ- 
ism. Study  this  organism  in  pure  cultures. 

The  same  may  be%  predicted  for  the  tube  which  has 
been  heated  for  ten  minutes,  though  the  colonies  will  be 
fewer  in  number. 

The  thirty-minute  tube  may  or  may  not  give  one  or 
two  colonies  of  the  same  organism. 

The  tube  which  has  been  heated  for  one  hour  is 
usually  sterile. 

The  bouillon  tubes  from'  the  first  and  second  tubes 
which  were  heated  will  usually  show  the  presence  of 
only  one  organism — the  bacillus  which  gave  rise  to  the 
pellicle-formation  in  the  original  mixture.  This  organ- 
ism is  bacillus  subtilis.  It  is  especially  adapted  to  the 
study  of  those  various  degrees  of  resistance  to  heat  that 
spore-forming  bacteria  exhibit  at  different  stages  of  their 
development. 

Inoculate  about  100  c.c.  of  sterilized  bouillon  with 
a  very  small  quantity  of  a  pure  culture  of  this  organism, 
and  allow  it  to  stand  in  a  warm  place  for  about  six 
hours.  Now  subject  this  culture  to  the  action  of  steam 
-for  five  minutes ;  it  will  be  seen  that  sterilization,  as  a 
rule,  is  complete. 

Treat  in  the  same  way  a  second  flask  of  bouillon, 
inoculated  in  the  same  way  with  the  same  organism, 
but  after  having  stood  in  a  warm  place  for  from  forty- 
eight  to  seventy-two  hours — that  is,  until  spores  have 
formed — and  it  will  be  found  that  sterilization  is  not 
complete  :  the  spores  of  this  organism  have  resisted  the 
action  of  steam  for  five  minutes. 

To  determine  if  sterilization  is  complete  always  resort 
to  the  culture  methods,  as  the  macroscopic  and  micro- 


652  BACTERIOLOGY. 

scopic  methods  are  deceptive ;  cloudiness  of  the  media 
or  the  presence  of  bacteria  microscopically  does  not 
always  signify  that  the  organisms  possess  the  property 
of  life. 

Inoculate  in  the  same  way  a  third  flask  of  bouillon 
with  a  very  small  drop  from  one  of  the  old  cultures  upon 
which  the  pellicle  has  formed  ;  mix  it  well  and  subject 
it  to  the  action  of  steam  for  two  minutes ;  then  place  it 
to  one  side  for  from  twenty  to  twenty-four  hours,  and 
again  heat  for  two  minutes  ;  allow  it  to  stand  for  another 
twenty-four  hours,  and  repeat  the  process  on  the  third 
day.  No  pellicle  will  be  formed,  and  yet  spores  were 
present  in  the  original  mixture,  and,  as  we  have  seen, 
the  spores  of  this  organism  are  not  killed  by  an  exposure 
of  five  minutes  to  steam.  How  can  this  result  be  ac- 
counted for  ? 

Saturate  several  pieces  of  cotton  thread,  each  about  2 
cm.  long,  in  the  original  decomposed  bouillon,  and  dry 
them  carefully  at  the  ordinary  temperature  of  the  room  ; 
then  at  a  little  higher  temperature — about  40°  C. — to 
complete  the  process.  Regulate  the  temperature  of  the 
hot-air  sterilizer  for  about  100°  C.,  and  subject  several 
pieces  of  this  infected  and  dried  thread  to  this  temper- 
ature for  the  same  lengths  of  time  that  we  exposed  the 
same  organisms  in  bouillon  to  the  steam,  viz.,  five,  ten, 
thirty,  and  sixty  minutes.  At  the  end  of  each  of  these 
periods  remove  a  bit  of  thread,  and  prepare  a  set  of 
plates  or  Esmarch  tubes  from  it.  Are  the  results  anal- 
ogous to  those  obtained  when  steam  was  employed  ? 

Increase  the  temperature  of  the  dry  sterilizer  and 
repeat  the  process.  Determine  the  temperature  and 
time  necessary  for  the  destruction  of  these  organisms 
by  dry  heat.  These  threads  should  not  be  simply 


STERILIZATION  BY  STEAM  AND   HOT  AIR.  653 

laid  upon  the  bottom  of  the  sterilizer,  but  should  be 
suspended  from  a  glass  rod,  which  may  be  placed  inside 
the  oven,  extending  across  its  top  from  side  to  side. 

Place  several  of  the  infected  threads  in  the  centre  of 
a  bundle  of  rags.  Subject  this  to  a  temperature  neces- 
sary to  sterilize  the  threads  by  the  dry  method.  Treat 
another  similar  bundle  to  sterilization  by  steam.  In 
what  way  do  the  results  of  the  two  processes  differ? 


CHAPTER   XXVIII. 

Methods  of  testing  disinfectants  and  antiseptics— Experiments  illus- 
trating the  precautions  to  be  taken — Experiments  in  skin-disin- 
fection. 

DETERMINATION   OF   DISINFECTANT   PROPERTIES. 

THERE  are  several  ways  of  determining  the  germicidal 
value  of  chemical  substances,  the  most  common  being 
to  expose  organisms  dried  upon  bits  of  silk  thread  to 
the  disinfectant  for  different  lengths  of  time,  and  then, 
after  removing,  and  carefully  washing  the  threads  in 
water,  to  place  them  in  nutrient  media  at  a  favorable 
temperature,  and  notice  if  any  growth  appears.  If  no 
growth  results,  the  disinfection  is  presumably  successful. 
Another  method  is  to  mix  fluid  cultures  of  bacteria  with 
the  disinfectant  in  varying  proportions,  and,  after  dif- 
ferent intervals  of  time,  to  determine  if  disinfection  is 
in  progress  by  transferring  a  portion  of  the  mixture  to 
nutrient  media,  just  as  in  the  other  methods  of  work. 

By  the  first  of  these  processes  the  bits  of  thread, 
usually  about  1  to  2  cm.  long,  are  placed  in  a  dry  test- 
tube  provided  with  a  cotton  plug  and  carefully  sterilized, 
either  by  the  dry  method  or  in  the  steam  sterilizer, 
before  using.  They  are  then  immersed  in  a  pure 
bouillon  culture  or  in  a  salt-solution  suspension  of  the 
organism  upon  which  the  disinfectant  is  to  be  tested. 
I  say  "  pure  culture/7  because  it  is  always  desirable  in 
testing  a  substance  to  determine  its  germicidal  value 

654 


METHODS  OF  TESTING  DISINFECTANTS.      655 

for  several  different  resistant  species  of  bacteria,  both 
in  the  vegetating  and  in  the  spore  stage,  and  also  be- 
cause it  is  only  by  the  use  of  pure  cultures  of  familiar 
species  that  it  is  possible  to  distinguish  between  the 
colonies  growing  from  the  individuals  that  have  not 
been  destroyed  by  the  disinfectant  under  investigation 
and  those  of  unknown  species  that  may  appear  upon 
the  plate  as  contaminations  occurring  during  the  manip- 
ulation. 

After  the  threads  have  remained  in  the  culture  or 
suspension  for  from  five  to  ten  minutes  they  are  re- 
moved under  antiseptic  precautions  and  carefully  sepa- 
rated and  spread  upon  the  bottom  of  a  sterilized  Petri 
dish,  which  is  then  placed  either  in  the  incubator  at  a 
temperature  not  exceeding  38°  C.  until  the  excess  of  fluid 
has  evaporated,  or  in  a  desiccator  over  sulphuric  acid, 
calcium  chloride,  or  any  other  drying-agent.  The  threads 
are  not  left  there  until  absolutely  dry,  but  only  until  the 
excess  of  moisture  has  evaporated.  When  sufficiently  dry 
they  are  immersed  in  solutions  of  the  disinfectant  of  dif- 
ferent but  known  strengths  for  a  fixed  interval  of  time, 
say  one  or  two  hours,  after  which  they  are  removed, 
rinsed  in  sterilized  distilled  water  to  remove  the  ex- 
cess of  disinfectant  adhering  to  them,  and  placed  in 
fresh,  sterile  culture-media,  which  are  then  placed  in 
the  incubator  at  from  37°  to  38°  C.  If  after  twenty- 
four  to  forty-eight  or  seventy-two  hours  a  growth  occurs 
at  or  about  the  bit  of  thread,  and  if  this  growth  consists 
of  the  organism  with  which  the  test  was  made,  mani- 
festly there  has  been  no  disinfection;  if  no  growth 
occurs  after,  at  most,  ninety-six  hours,  it  is  safe  to  pre- 
sume that  the  bacteria  have  been  killed,  unless  our 
efforts  at  rinsing  off  the  excess  of  disinfectant  from 


656  B  A  CTERIOL  OGY. 

the  thread  have  not  been  successful,  and  a  small  amount 
of  disinfectant  is  still  active  in  preventing  development 
— i.  e.,  is  acting  as  an  antiseptic. 

By  the  process  in  which  cultures  or  suspensions 
of  the  organisms  are  mixed  with  different  but  known 
strengths  of  the  disinfectant  a  small  portion  of  the 
mixture,  usually  a  loopful  or  a  drop,  is  transferred 
at  the  end  of  a  definite  time  to  the  fresh  medium 
which  is  to  determine  whether  the  organisms  have 
been  killed  or  not.  This  is  commonly  a  tube  of  fluid 
agar-agar,  which  is  poured  into  a  Petri  dish,  allowed  to 
solidify,  and  placed  in  the  incubator,  as  in  the  preceding 
method. 

After  the  minimum  strength  of  disinfectant  necessary 
to  destroy  the  vitality  of  the  organisms  with  which  we 
are  working  has  been  determined  for  any  fixed  time,  it 
remains  for  us  to  decide  what  is  the  shortest  time  in  which 
this  strength  will  have  the  same  effect.  We  then  work 
with  a  constant  dilution  of  the  disinfectant,  but  with 
varying  intervals  of  exposure — one,  five,  ten  minutes, 
etc. — until  we  have  decided  not  only  the  minimum 
amount  of  disinfectant  required  for  the  destruction  of 
the  bacteria,  but  the  shortest  time  necessary  for  this 
under  known  conditions. 

A  factor  not  to  be  lost  sight  of  is  the  temperature 
at  which  these  experiments  are  conducted,  for  it 
must  always  be  borne  in  mind  that  the  action  of  a  dis- 
infectant is  usually  more  energetic  at  a  higher  than  at  a 
lower  temperature. 

Now  in  both  of  these  methods  it  is  easy  to  see  that 
unless  special  precautions  are  taken  a  minute  portion  of 
the  disinfectant  may  be  carried  along  with  the  thread, 
or  drop,  into  the  medium  which  is  to  determine  whether 


METHODS  OF  TESTING  DISINFECTANTS.      657 

the  organisms  do  or  do  not  possess  the  power  of  growth, 
and  there  have  a  restraining  or  antiseptic  action.  For 
organisms  in  their  normal  condition — that  is,  those 
which  have  never  been  exposed  to  the  action  of  a  dis- 
infectant— the  amount  of  certain  disinfectants  that  is 
necessary  to  restrain  growth  is  very  small  indeed ;  and 
for  organisms  that  have  already  been  exposed  for  a 
time  to  such  agents  this  amount  is  very  much  less 
It  is  plain,  then,  that  if  the  test  is  to  be  an  accurate 
one,  precautions  must  be  taken  against  admitting  this 
minute  trace  of  disinfectant  to  the  medium  with  which 
we  are  to  determine  whether  the  bacteria  exposed  to  the 
disinfectant  were  killed  or  not.  The  precautions  hitherto 
taken  to  prevent  this  accident  have  been,  when  the  threads 
were  employed,  washing  them  in  sterilized  distilled 
water  and  then  in  alcohol ;  or,  where  fluid  cultures  were 
mixed  with  the  disinfectant  in  solution,  an  effort  was 
usually  made  to  dilute  the  amount  of  disinfectant  car- 
ried over,  to  a  point  at  which  it  lost  its  inhibiting 
power. 

While  such  precautions  are  sufficient  in  many  cases, 
they  do  not  answer  for  all.  Certain  chemicals  have  the 
property  of  combining  so  firmly  with  the  threads  upon 
which  the  bacteria  are  located  as  to  require  other  special 
means  of  ridding  the  threads  of  them  ;  and  in  solutions 
in  which  proteid  substances  are  present  along  with  the 
bacteria  a  similar  union  between  them  and  the  disin- 
fectant may  likewise  take  place.  In  both  instances  this 
amount  of  disinfectant  adhering  to  the  threads  or  in 
combination  with  the  proteids  must  be  gotten  rid  of, 
otherwise  the  results  of  the  test  may  be  fallacious.  A  par- 
tial solution  of  the  problem  is  given  through  studies  that 
have  been  made  upon  corrosive  sublimate  in  its  various 

42 


658  BA  CTERIOLOG  Y. 

applications  for  disinfecting  purposes,  and  in  this  con- 
nection it  has  been  shown  by  Shaefer }  that  it  is  impos- 
sible to  rid  silk  threads  of  the  corrosive  sublimate  ad- 
hering to  them  by  simple  washing,  as  the  sublimate 
acts  as  a  mordant  and  forms  a  firm  union  with  the  tis- 
sues of  the  threads.  Braatz  2  found  the  same  to  hold 
good  for  catgut.  For  example,  he  found  that  catgut  which 
had  been  immersed  in  solutions  of  corrosive  sublimate 
gave  the  characteristic  reactions  of  the  salt  after  having 
been  immersed  for  five  weeks  in  distilled  water  which 
had  been  repeatedly  renewed.  Braatz  remarks  that 
a  similar  combination  between  sublimate  and  cotton 
will  take  place  after  a  long  time  ;  but  it  occurs  so 
slowly  that  it  cannot  interfere  with  disinfection-experi- 
ments in  the  same  way  that  silk  does. 

The  most  successful  attempt  at  removing  all  traces 
of  sublimate  from  the  threads  or  from  the  proteid  sub- 
stances in  which  are  located  the  bacteria  whose  vitality 
is  to  be  tested  was  made  by  Geppert,  who  subjected 
them  to  the  action  of  ammonium  sulphide  in  solution. 
By  this  procedure  the  mercury  is  converted  into  inert, 
insoluble  sulphide,  and  has  no  inhibiting  effect  upon  the 
growth  of  those  bacteria  that  did  not  succumb  to  its 
action  when  in  the  form  of  the  bichloride. 

In  the  second  method  of  testing  disinfectants  men- 
tioned above — that  is,  when  cultures  of  bacteria  and 
solutions  of  the  disinfectant  are  mixed,  and  after  a  time 
a  drop  of  the  mixture  is  removed  and  added  to  sterile 
nutrient  media — the  inhibiting  amount  of  disinfectant 
can  readily  be  gotten  rid  of  by  dilution ;  that  is  to  say, 

1  Shaefer  :  Berliner  klin.  Wochenschrift,  1890,  No.  3,  p.  50. 

2  Braatz  :  Centralblatt  f iir  Bakteriolgie  und  Parasitenkunde,  Bd .  vii. 
tfo.  1,  p.  8. 


METHODS  OF  TESTING  DISINFECTANTS.      651) 

instead  of  transferring  the  drop  directly  to  the  fresh 
medium,  add  it  to  10  or  12  c.c.  of  sterilized  salt-solu- 
tion (0.6-0.7  per  cent,  of  NaCI  in  distilled  water)  or 
distilled  water,  and  after  thoroughly  shaking  add  a  drop 
of  this  to  the  medium  in  which  the  power  of  develop- 
ment of  the  bacteria  is  to  be  determined. 

Another  important  point  to  be  borne  in  mind  in 
testing  disinfectants  is  the  necessity  of  so  adjusting  the 
conditions  that  each  individual  organism  will  be  ex- 
posed to  the  action  of  the  agent  used.  When  clumps 
of  bacteria  exist  we  are  not  always  assured  of  this,  for 
only  those  on  the  surface  of  the  clump  may  be  affected, 
while  those  in  the  centre  of  the  mass  may  escape,  being 
protected  by  those  surrounding  them.  These  clumps 
and  minute  masses  are  especially  liable  to  be  present 
in  fluid  cultures  and  in  suspensions  of  bacteria,  and 
must  be  eliminated  before  the  test  is  begun,  if  this  is  to 
be  made  by  mixing  them  with  solutions  of  the  agent  to 
be  tested.  This  is  best  accomplished  in  the  following 
way  :  the  organisms  should  be  cultivated  in  bouillon 
containing  sand  or  finely  divided  particles  of  glass; 
after  growing  for  a  sufficient  length  of  time  they  are 
to  be  shaken  thoroughly,  in  order  that  all  clumps  may 
be  mechanically  broken  up  by  the  sand.  The  culture  is 
then  filtered  through  a  tube  containing  closely  packed 
glass-wool . 

The  filtration  may  be  accomplished  without  fear  of 
contamination  of  the  culture  by  the  employment  of  an 
Allihin  tube,  which  is  practically  a  thick-walled  test- 
tube  drawn  out  to  a  finer  tube  at  its  blunt  end  so  as  to 
convert  it  into  a  sort  of  cylindrical  funnel.  The  tube 
when  ready  for  use  has  the  appearance  shown  in  Fig. 
111. 


660 


BACTERIOLOGY. 


FIG.  100. 


This  tube,  after  being  plugged  at  the  bottom  with 
glass-wool  (a,  Fig.  100),  and  at  its  wide  extremity 
with  cotton-wool,  is  placed  vertically, 
small  end  down,  into  an  Erlenmeyer 
flask  of  about  100  c.c.  capacity  and 
sterilized  in  a  steam  sterilizer  for  the 
proper  time.  It  is  kept  in  the  sterilizer 
until  it  is  to  be  used,  which  should  be 
as  soon  as  possible  after  sterilization. 

The  watery  suspension  or  bouillon 
culture  of  the  organisms  is  now  to  be 
filtered  repeatedly  through  the  glass- 
wool  into  sterilized  flasks  until  a  de- 
gree of  transparency  is  reached  which 
will  permit  the  reading  of  moderately 
fine  print  through  a  layer  of  the  fluid 
about  2  cm.  thick — i.  e.,  through  an  or- 
dinary test-tube  full  of  it.  This  filtrate 
can  then  be  subjected  to  the  action  of 
the  disinfectant.  As  a  rule,  the  results 
are  more  uniform  than  when  no  atten- 
tion is  paid  to  the  presence  of  clumps. 
It  is  scarcely  necessary  to  say  that  in 
the  practical  employment  of  disinfec- 
tants outside  the  laboratory  no  such  pre- 
cautions are  taken ;  but  in  laboratory 
work,  where  it  is  desired  to  determine 
exactly  the  value  of  different  substances 
as  germicides,  all  the  precautions  men- 
tioned will  be  found  essential  to  preci- 
sion. 

The  disinfectant  value  of  gases  and  vapors   is   de- 
termined  by  their  action  upon   test-objects   in   closed 


Cylindrical  fun- 
nel used  for  filtering 
cultures  on  which 
disinfectants  are  to 
be  tested. 


METHODS  OF  TESTING  DISINFECTANTS.      GUI 

chambers.  The  object  is  to  determine  the  proportion 
of  the  gas,  when  mixed  with  air,  that  is  required  to 
destroy  the  bacteria  exposed  to  its  action  in  a  given 
time.  For  this  purpose  the  test  is  usually  made  as 
follows  :  under  a  sterilized  bell-glass  of  known  capacity 
the  test-objects  are  placed.  Into  the  chamber  is  then 
admitted  sufficient  of  a  mixture  of  air  and  the  gas  under 
consideration,  of  known  proportions,  to  displace  com- 
pletely all  the  air ;  or  the  pure  gas  itself  may  be  intro- 
duced in  amount  necessary  to  give  the  desired  dilution 
when  mixed  with  the  air  in  the  chamber.  At  the 
expiration  of  the  time  decided  upon  for  the  test  the 
infected  articles  are  removed  and  the  vitality  of  the 
bacteria  upon  them  is  determined. 

In  the  case  of  vapors  of  volatile  fluids,  such,  for 
instance,  as  formalin,  the  fluid  is  placed  under  the  bell- 
glass  in  an  open  dish ;  in  another  open  dish  the  test- 
objects  are  placed.  The  bell-glass  is  then  sealed  to  an 
underlying  ground-glass  plate  by  vaselin  or  paraffin, 
and  the  fluid  is  allowed  to  vaporize  at  ordinary  room- 
temperature.  The  point  here  to  be  decided  is  the  vol- 
ume cr  weight  of  such  a  fluid  that  it  is  necessary  to 
expose  in  an  air-chamber  of  known  cubic  capacity  in 
order  that  bacteria  may  be  destroyed  by  its  vapor  in  a 
given  time. 

In  determining  the  germicidal  value  of  different 
chemical  agents  for  certain  pathogenic  bacteria  sus- 
ceptible animals  are  sometimes  inoculated  with  the 
organisms  after  they  have  been  exposed  to  the  disin- 
fectant. If  no  pathological  condition  results,  disinfec- 
tion is  presumed  to  have  been  successful ;  while  if  the 
condition  characteristic  of  the  activities  of  the  given 
organism  in  the  tissues  of  this  animal  appears,  the 


662  BACTERIOLOGY. 

reverse  is  the  case.  The  objections  to  this  method 
are  :  "  First.  The  test-organisms  may  be  modified  as 
regards  reproductive  activity  without  being  killed ; 
and  in  this  case  a  modified  form  of  the  disease  may 
result  from  the  inoculation,  of  so  mild  a  character 
as  to  escape  observation.  Second.  An  animal  that 
has  suffered  this  modified  form  of  the  disease  enjoys 
protection,  more  or  less  perfect,  from  future  attacks, 
and  if  used  for  a  subsequent  experiment  may,  by 
its  immunity  from  the  effects  of  the  pathogenic  test- 
organism,  give  rise  to  the  mistaken  assumption  that 
this  had  been  destroyed  by  the  action  of  the  germicidal 
agent  to  which  it  had  been  subjected."  (Sternberg.) 

DETERMINATION    OF    ANTISEPTIC    PROPERTIES. 

For  this  purpose  sterile  media  are  employed,  and  are 
usually  arranged  in  two  groups :  the  one  to  remain 
normal  in  composition  and  to  serve  as  controls,  while  to 
the  other  the  substance  to  be  tested  is  to  be  added  in  dif- 
ferent but  known  strengths.  It  is  customary  to  employ 
test-tubes  each  containing  an  exact  amount  of  bouillon, 
gelatin,  or  agar-agar,  as  the  case  may  be.  To  each  tube 
a  definite  amount  of  the  antiseptic  is  added,  and  if  it 
is  not  of  a  volatile  nature  or  not  injured  by  heat,  the 
tubes  may  then  be  sterilized.  After  this  they  are  to  be 
inoculated  with  the  organism  with  which  the  test  is  to 
be  made,  and  at  the  same  time  one  of  the  "  control  "- 
tubes  (one  of  those  to  which  no  antiseptic  has  been 
added)  is  inoculated.  They  are  all  then  to  be  placed 
in  the  incubator  and  kept  under  observation.  If  at  the 
end  of  twenty-four,  forty-eight,  or  seventy-two  hours 
no  growth  appears  in  any  but  the  "  control  "-tubes,  it 
is  evident  that  the  antiseptic  must  be  added  in  smaller 


EXPERIMENTS.  663 

amounts,  for  we  are  to  determine  the  point  at  which  it 
/.s  not  as  well  as  that  at  which  it  is  capable  of  preventing 
development.  The  experiment  is  then  repeated,  using 
smaller  amounts  of  the  antiseptic  until  we  reach  a  point 
at  which  growth  just  occurs,  notwithstanding  the  pres- 
ence of  the  antiseptic  ;  the  amount  necessary  for  antisep- 
sis is  then  a  trifle  greater  than  that  used  in  the  last  tube. 
If,  for  example,  there  was  no  development  in  the  tubes 
in  which  the  antiseptic  was  present  in  the  proportion 
of  1  :  1000,  and  growth  in  the  one  in  which  it  was 
present  in  1  :  1400,  the  experiment  should  be  repeated 
with  strengths  of  the  antiseptic  corresponding  to 
1  : 1000,  1  : 1100,  1  :  1200,  1  : 1300,  1  : 1400,  and  in 
this  way  one  ultimately  determines  the  amount  by  which 
growth  is  just  prevented  ;  this  represents  the  antiseptic 
value  of  the  substance  for  the  organism  with  which  it 
was  tested. 

EXPERIMENTS. 

To  each  of  three  tubes  containing  10  c.c. — one  of 
physiological  salt-solution,  another  of  bouillon,  a  third 
of  fluid  blood-serum — add  as  much  of  a  culture  of 
micrococcus  aureus  as  can  be  held  upon  a  looped 
platinum  wire.  Break  this  up  carefully  to  eliminate 
clumps,  and  then  add  exactly  10  c.c.  of  a  1  :  500  solution 
of  corrosive  sublimate.  Mix  thoroughly,  and  at  the 
end  of  three  minutes  transfer  a  drop  from  each  tube 
into  tubes  of  liquefied  agar-agar,  and  pour  these  into 
Petri  dishes.  Label  each  dish  carefully  and  place  them 
in  the  incubator.  Are  the  results  the  same  in  all  the 
plates  ?  How  are  the  differences  to  be  explained  ?  To 
what  strength  of  the  disinfectant  were  the  organisms 
exposed  in  the  experiment? 


664  B  A  CTERIOLOG  Y. 

To  each  of  two  tubes — the  one  containing  10  c.c. 
of  normal  salt-solution,  the  other  of  bouillon — add  as 
much  of  a  spore-containing  culture  of  anthrax  bacilli 
as  can  be  held  upon  a  loop  of  platinum  wire.  Dis- 
tribute this  uniformly  through  the  medium,  and  then 
add  exactly  10  c.c.  of  a  1  :  500  solution  of  corrosive 
sublimate.  Mix  thoroughly,  and  at  the  end  of  five 
minutes  transfer  a  drop  from  each  tube  to  tubes  of 
liquefied  agar-agar.  Pour  these  immediately  into 
Petri  dishes.  Label  each  dish  carefully  and  place  them 
in  the  incubator.  Note  the  results  at  the  end  of  twenty- 
four,  forty-eight,  and  seventy-two  hours.  How  do 
you  explain  them  ? 

Make  identically  the  same  experiment  with  the  same 
spore-containing  culture  of  anthrax  bacilli,  except  that 
the  drop  from  the  mixture  is  to  be  transferred  to  10  c.c. 
of  a  mixture  of  equal  parts  of  ammonium  sulphide  and 
sterilized  distilled  water.  After  remaining  in  this  for 
about  half  a  minute,  a  drop  is  to  be  transferred  to  a 
tube  of  liquefied  agar-agar,  poured  into  Petri  dishes, 
labelled,  and  placed  in  the  incubator.  Note  the  results. 
Do  they  correspond  with  those  obtained  in  the  pre- 
ceding experiment?  How  are  the  differences  ex- 
plained ? 

Prepare  a  1  : 1000  solution  of  corrosive  sublimate. 
To  each  of  twelve  tubes  containing  exactly  10  c.c.  of 
bouillon  add  one  drop  to  the  first,  two  drops  to  the 
second,  and  so  on  until  the  last  tube  has  had  twelve 
drops  added  to  it.  Mix  thoroughly  and  then  inoculate 
each  with  one  wire-loopful  of  a  bouillon  culture  of 
micrococcus  aureus.  Place  them  all  in  the  incubator 


EXPERIMENTS.  665 

after  carefully  labelling  them.    Note  the  order  in  which 
growth  appears. 

Do  the  same  with  anthrax  spores,  with  spores  of 
bacillus  subtilis,  and  with  the  typhoid  bacillus,  and  com- 
pare the  results.  From  these  experiments,  what  will  be 
the  strength  of  corrosive  sublimate  necessary  to  anti- 
sepsis under  these  conditions  for  the  organisms  em- 
ployed ? 

Make  a  similar  series  of  experiments  using  a  5  per 
cent,  solution  of  carbolic  acid. 

Determine  the  antiseptic  value  of  the  common  disin- 
fectants for  the  organisms  with  which  you  are  working. 

Determine  the  time  necessary  for  the  destruction  of 
the  organisms  with  which  you  are  working,  by  corro- 
sive sublimate  in  1  : 1000  solution,  under  different  con- 
ditions— with  and  without  the  presence  of  albuminous 
bodies  other  than  the  bacteria,  and  under  varying  con- 
ditions of  temperature. 

In  making  these  experiments  be  careful  to  guard 
against  the  introduction  of  sufficient  sublimate  into  the 
agar-agar  with  which  the  Petri  plate  is  to  be  made  to 
inhibit  the  growth  of  the  organisms  which  may  not  have 
been  destroyed  by  the  sublimate.  This  may  be  done  by 
transferring  two  drops  from  the  mixture  of  sublimate 
and  organism  into  not  less  than  10  c.c.  of  sterilized 
physiological  salt-solution,  in  which  they  may  be  thor- 
oughly shaken  for  from  one  to  two  minutes,  or  into  the 
solution  of  ammonium  sulphide  of  the  strength  given. 

To  10  c.c.  of  a  bouillon  culture  of  micrococcus 
aurcm  or  anthrax  spores  add  10  c.c.  of  a  1  : 500  solu- 


666  BACTERIOLOGY. 

tion  of  corrosive  sublimate,  and  allow  it  to  remain  in 
contact  with  the  organisms  for  only  one-half  the  time 
necessary  to  destroy  them  (use  an  organism  for  which 
this  has  been  determined).  Then  transfer  a  drop  of 
the  mixture  to  each  of  three  liquefied  agar-agar  tubes 
and  pour  them  into  Petri  dishes.  Place  them  in  the 
incubator  and  observe  them  for  twenty-four,  forty- 
eight,  and  seventy-two  hours.  No  growth  occurs.  How 
is  this  to  be  accounted  for  ? 

At  the  end  of  seventy-two  hours  inoculate  all  of  these 
plates  with  a  culture  of  the  same  organism  which  has 
not  been  exposed  to  sublimate,  by  taking  up  bits  of  cult- 
ure on  a  needle  and  drawing  it  across  the  plates.  A 
growth  now  results.  We  have  here  an  experiment  in 
which  organisms  which  have  been  exposed  to  sublimate 
for  a  much  shorter  time  than  necessary  to  destroy  them, 
when  transferred  directly  to  a  favorable  culture-medium 
do  not  grow,  and  yet,  when  the  same  organism  which 
has  not  been  exposed  to  sublimate  at  all  is  planted  upon 
the  same  medium  it  does  grow.  How  is  this  to  be  ac- 
counted for? 

SKIN-DISINFECTION. — With  a  sterilized  knife  scrape 
from  the  skin  of  the  hands,  at  the  root  of  the  nails,  and 
under  the  nails,  small  particles  of  epidermis.  Prepare 
plates  from  them.  Note  the  results. 

Wash  the  hands  carefully  for  ten  minutes  in  hot 
water  and  scrub  them  during  this  time  with  soap  and 
a  sterilized  brush.  Rinse  them  in  hot  water.  Again 
prepare  plates  from  scrapings  of  the  skin  on  the  fingers, 
at  the  root  of  the  nails,  and  under  the  nails.  Note  the 
results. 

Again  wash  as  before  in  hot  water  with   soap  and 


EXPERIMENTS.  667 

brush,  rinse  in  hot  water,  then  soak  the  hands  for  five 
minutes  in  1  : 1000  corrosive  sublimate  solution,  and, 
as  before,  prepare  plates  from  scrapings  from  the  same 
localities.  Note  the  results. 

Repeat  this  latter  procedure  in  exactly  the  same  way, 
but  before  taking  the  scrapings  let  some  one  pour  am- 
monium sulphide  over  the  points  from  which  the  scrap- 
ings are  to  be  made.  After  it  has  been  on  the  hands 
about  three  minutes  again  scrape,  and  note  the  result 
upon  plates  made  from  the  scrapings. 

Wash  as  before  in  hot  water  and  soap,  rinse  in  clean 
hot  water,  immerse  for  a  minute  or  two  in  alcohol, 
after  this  in  1  : 1000  sublimate  solution,  and  finally  in 
ammonium  sulphide,  and  then  prepare  plates  from 
scrapings  from  the  points  mentioned. 

In  what  way  do  the  results  of  these  experiments 
differ  from  one  another? 

To  what  are  these  differences  due  ? 

What  have  these  experiments  taught? 

In  making  the  above  experiments  it  must  be  remem- 
bered that  the  strictest  care  is  necessary  in  order  to 
prevent  the  access  of  germs  from  without  into  our 
media.  The  hand  upon  which  the  experiment  is  being 
performed  must  be  held  away  from  the  body  and  must 
not  touch  any  object  not  concerned  in  the  experiment. 
The  scraping  should  be  done  with  the  point  of  a  knife 
that  has  been  sterilized  in  a  flame  and  allowed  to  cool. 
The  scrapings  may  be  transferred  directly  from  the 
knife-point  to  the  gelatin  by  means  of  a  sterilized  plat- 
inum wire  loop. 

The  brush  used  should  be  thoroughly  cleansed  and 
always  kept  in  1  :  1000  solution  of  corrosive  sublimate. 
It  should  be  washed  in  hot  water  before  using. 


APPENDIX. 


LIST  of  apparatus  and  materials  required  in  a  begin- 
ner's bacteriological  laboratory : 

MICROSCOPE   AND   ACCESSORIES. 

Microscope  with  coarse  and  fine  adjustment  and 
heavy,  firm  base ;  Abbe  sub-stage  condensing  system, 
arranged  either  as  the  "  simple  "  or  as  the  regular  Abbe 
condenser,  in  either  case  to  be  provided  with  iris  dia- 
phragm ;  objectives  equivalent,  in  the  English  nomen- 
clature, to  about  one-fourth  inch  and  one-sixth  inch 
dry,  and  one-twelfth  inch  oil-immersion  system;  a 
triple  revolving  nose-piece ;  three  oculars,  varying  in 
magnifying  power;  and  a  bottle  of  immersion  oil. 

Glass  slides,  English  shape  and  size  and  of  colorless 
glass. 

Six  slides  with  depressions  of  about  1  cm.  in  diameter 
in  centre. 

Cover-slips,  15  by  15  mm.  square  and  not  more  than 
from  0.15  to  0.18  mm.  thick. 

Forceps.  One  pair  of  fine-pointed  forceps  and  one 
pair  of  the  Cornet  or  Stewart  pattern,  for  holding  cover- 
slips. 

Platinum  needles  with  glass  handles.  One  straight, 
iiboiit  4  cm.  long;  one  looped  at  the  end,  about  4  cm. 
long;  and  one  straight,  about  8  cm.  long.  Glass 


670  APPENDIX. 

handles  to  be  about  3  mm.  in  thickness  and  from  15 
to  17  cm.  long. 

STAINING-    AND    MOUNTING-REAGENTS. 

200  c.c.  of  saturated  alcoholic  solution  of  fuchsin. 

200  c.c.  of  saturated  alcoholic  solution  of  gentian- 
violet. 

200  c.c.  of  saturated  alcoholic  solution  of  methylene- 
blue. 

200  grammes  of  pure  aniline. 

200  grammes  of  C.  P.  carbolic  acid. 

500  grammes  of  C.  P.  nitric  acid. 

500  grammes  of  C.  P.  sulphuric  acid. 

200  grammes  of  C.  P.  glacial  acetic  acid. 

1  litre  of  ordinary  93-95  per  cent,  alcohol. 

1  litre  of  absolute  alcohol. 
500  grammes  of  ether. 

500  grammes  of  pure  xylol. 

50  grammes  of  Canada  balsam  dissolved  in  xylol. 
100  grammes  of  Schering's  celloidin. 
10  grammes  of  iodine  and  30  grammes  of  potassium 
iodide  in  substance. 

100  grammes  of  tannic  acid. 
100  grammes  of  ferrous  sulphate. 
Distilled  water. 

FOR   NUTRIENT   MEDIA. 

^  pound  of  Liebig's  or  Armour's  beef-extract. 
250  grammes  of  Witte's  or  Sargent's  peptone. 

2  kilogrammes  of  gold-label  gelatin  (Hesteberg's). 
1 00  grammes  of  agar-agar  in  substance. 

200  grammes  of  sodium  chloride  (ordinary  table-salt). 
500  grammes  of  pure  glycerin. 


APPENDIX.  671 

50  grammes  of  pure  glucose. 

20  grammes  of  pure  lactose. 

100  grammes  of  caustic  potash. 

200  c.c.  of  litmus  tincture. 

10  grammes  of  rosolic  acid  (corallin). 

Blue  and  red  litmus-paper ;  curcuma  paper. 

5  grammes  of  phenolphtalein  in  substance. 

Filter-paper,  the  quality  ordinarily  used  by  druggists. 

100  grammes  of  pyrogallic  acid. 

1  kilogramme  of  C.  P.  granulated  zinc. 


GLASSWARE. 

200  best  quality  test-tubes,  slightly  heavier  than  those 
used  for  chemical  work,  about  12  to  13  cm.  long  and  12 
to  14  mm.  inside  diameter. 

15  Petri  double  dishes  about  8  or  9  cm.  in  diameter 
and  from  1  to  1.5  cm.  deep. 

6  Florence  flasks,  Bohemian  glass,  1000  c.c.  capacity. 

6  Florence  flasks,  Bohemian  glass,  500  c.c.  capacity. 

12  Erlenmeyer  flasks,  Bohemian  glass,  100  c.c. 
capacity. 

1  graduated  measuring-cylinder,  1000  c.c.  capacity. 

1  graduated  measuring-cylinder,  100  c.c.  capacity. 

25  bottles,  125  c.c.  capacity,  narrow  necks  with 
ground-glass  stoppers. 

25  bottles,  125  c.c.  capacity,  wide  mouths,  with 
ground-glass  stoppers. 

1  anatomical  or  preserving  jar,  with  tightly  fitting 
cover,  of  about  4  litres  capacity,  for  collecting  blood- 
serum. 

2  battery  jars  of  about   2  litres  capacity,  provided 
with  loosely  fitting,  weighted,  wire-net  covers  for  mice. 


672  APPENDIX. 

10  feet  of  soft-glass  tubing,  2  or  3  mm.  inside  diam- 
eter. 

20  feet  of  soft-glass  tubing,  4  mm.  inside  diameter. 

6  glass  rods,  18  to  20  cm.  long  and  3  or  4  mm.  in 
diameter. 

6  pipettes  of  1  c.c.  each,  divided  into  tenths. 

2  pipettes  of  10  c.c.  each,  divided  into  cubic  centi- 
metres and  fractions. 

1  burette  of  50  c.c.  capacity,  divided  into  cubic  cen- 
timetres and  fractions. 

1  separating-funnel  of  750  c.c.  capacity  for  filling 
tubes. 

2  glass  funnels,  best  quality,  about  15  cm.  in  diam- 
eter. 

2  glass  funnels,  best  quality,  about  8  cm.  in  diameter. 
2  glass  funnels,  best  quality,  about   4  or  5   cm.  in 
diameter. 

2  porcelain  dishes,  200  c.c.  capacity. 

6  ordinary  water  tumblers  for  holding  test-tubes. 

1  ruled  plate  for  counting  colonies. 

1  gas-generator,  600  c.c.  capacity,  pattern  of  Kipp 
or  v.  Wartha. 

BURNERS,   TUBING,   ETC. 

2  Bunsen  burners,  single  flame'. 
1  Rose-burner. 

1  Koch  safety-burner,  single  flame. 
6  feet  of  white-rubber  gas-tubing. 
12  feet  of  pure  red-rubber  tubing,  5  to  6  mm.  inside 
diameter. 

1  ther mo-regulator,  pattern  of  L.  Meyer  or  Reich ert. 

2  thermometers,  graduated  in  degrees  of  Centigrade, 
registering  from  0  to  100°  0.,  graduated  on  the  stem. 


APPENDIX.  673 

1  thermometer  graduated  in  tenths  and  registering 
from  0  to  50°  C. 

1  thermometer  registering  to  200°  C. 

INSTRUMENTS,    ETC. 

1  microtome,  pattern  of  Schanze,  with  knife. 

1  razor-strop. 

6  cheap-quality  scalpels,  assorted  sizes. 

2  pairs  heavy  dissecting-forceps. 

1  pair  medium-size  straight  scissors. 
1  pair  small-size  straight  scissors. 

1  hypodermic  syringe  that  will  stand  steam  steriliza- 
tion. 

2  teasing-needles. 

1  pair  long-handled  crucible-tongs  for  holding  mice. 

1  wire  mouse-holder. 

2  small  pine  boards  on  which  to  tack  animals  for 
autopsy. 

2  covered  stone  jars  for  disinfectants  and  for  receiv- 
ing infected  materials. 

INCUBATORS    AND    STERILIZERS. 

1  incubator,  simple  square  form,  either  entirely  of 
copper  or  of  galvanized  iron  with  copper  bottom. 

1  medium-size  hot-air  sterilizer  with  double  walls, 
asbestos  jacket,  and  movable  false  bottom  of  copper 
plates. 

1  medium-size  steam  sterilizer ;  either  the  pattern  of 
Koch  or  that  known  as  the  Arnold  steam  sterilizer, 
preferably  the  latter. 

MISCELLANEOUS. 

1  pair  of  balances,  capacity  1  kilogramme ;  accurate 
to  0.2  gramme. 

43 


674  APPENDIX. 

1  set  of  cork-borers. 
1  hand-lens. 

1  wooden  filter-stand. 

2  iron  stands  with  rings  and  clamps. 

3  round,  galvanized  iron-wire  baskets  to  fit  loosely 
into  steam  sterilizer. 

3  square,  galvanized  iron-wire  baskets  to  fit  loosely 
into  hot-air  sterilizer. 

1  sheet-iron  box  for  sterilizing  pipettes,  etc. 

1  covered  agate-ware  saucepan,  1200  c.c.  capacity. 

2  iron  tripods. 

1  yard  of  moderately  heavy  wire  gauze. 

2  test-tube  racks,  each  holding  24  tubes,  12  in  a  row. 

1  constant-level,  cast-iron  water-bath. 

2  potato-knives. 

2  test-tube  brushes  with  reed  or  wire  handles. 

Cotton-batting. 

Copper  wire,  wire  nippers. 

Round  and  triangular  files. 

Labels. 

Towels  and  sponges. 


INDEX. 


ABBE,  30 
substage   condensing   system 

of,  203 

Abbott  and  Gildersleeve,  37 
Abscess,  histological  study  of,  276 

production  of,  275 
Abscess-wall,  278 
Acid-proof  bacteria,  365 
Acids,  production  of,  40 
Actinomyces  bovis,  378 
Eppingeri,  386 
farcinicus,  385 
madurse,  382 
pseudotuberculosis,  388 
Actinomycetes,  376 
Aerobic  bacteria,  44 
Aerobioscope,  644 
Agar-agar,  cultures  on,  195 
preparation  of  (see  Media), 
properties  of,  102 
Agglutinins,  431,  436,  578 
Air,  bacteriological  analysis  of,  640 

640 

Petri's  method,  641 
Sedgwick-Tucker    method, 

642 

Alexin  theory  of  Buchner,  583 
Alexins,  573,  583 
Alkali,  production  of,  40 
Alkaloids,  vegetable,  41 
Ammonia,  test  for,  232 
Anaerobic  bacteria,  44 

methods  of  cultivating,  220- 

226 

Buchner's,  222 
Esmarch's,  226 
Franker s,  223 
Hesse's,  221 

Kitasato  and  Weil's,  225 
Koch's,  220 
Liborins's,  221 
Park's,  226 


Aniline  dyes  for    differentiating 

bacteria,  214 
Animals,   fluctuations    in   weight 

and  temperature  of,  250 
inoculation  of,  235 

apparatus  used  in,  237-241 
intralymphatic,  247 
intraocular,  250 
intraperitoneal   and    pleural, 

248 

intravascular,  242 
subcutaneous,  235 
observations    of,  after  inocula- 

lation,  250 

postmortem  examination  of,  258 
cultures  from  tissues  of,  260 
disinfection  of  implements 

after,  262 
disposal  of   remains  from, 

262 

incision  through  skin,  258 
Nuttall's  spear  for  use  at, 

261 
opening  the  body  cavities, 

259 

position  of  animal,  258 
precautions  during,  258 
preservation  of  tissues  after, 

262 
Anthrax,  510 

animals  susceptible  to,  517 
bacterium  of,  510 
biology  of,  513 
discovery  of,  510 
experiments  with,  521 
immune  serum,  526 
morphology  of,  510 
pathogenesis  of,  516 
protective  inoculation  against, 

518 

spore  formation  of,  511 
staining  of,  515 

675 


676 


INDP;X. 


Anthrax,   bacterium   of,  sympto- 
matic bacillus  of,  549 
vaccines  of,  519 
Antidysenteria  serum,  471 
Antilysin,  279 
Antiplague  serum,  323 
Antiseptic,  definition  of,  89 
Antiseptics,  mode  of  action  of,  89 

tests  of,  662 

Antistaphylococcus  serum,  279 
Antistreptococcus  serum,  287 
Antitoxin,  diphtheria,  422 

tetanus,  542 
Apparatus     for      bacteriological 

work,  669 
preparation  of,  138 
Appendix,  list  of  apparatus,  669 
Arloing,   Cornevin   and  Thomas, 

549 

Arnold  steam  sterilizer,  83 
Aronson,  288 
Auerbach,  38 
Autoclave,  84 

BACILLI,  58 
differentiation  from  spores,  64 
flagella  upon,  66 
involution-forms  of,  60 
life-cycle  of,  59 
mode  of  multiplication  of,  57, 

59 

motility  of,  66 
spore-formation  in,  63 
Bacillus,  55 
anthracis,  510 
butter,  367 
Chauvei,  549 
coli,  54,  291,  453 

characteristics,    cultural,    of, 

455 

morphological,  455 
pathogenic,  458 
differentiation  of,  from  bacil- 
lus typhosus,  457 
where  found,  453 
"  comma,"  473 
diphtherise,  399 
dysenterise,  464 
influenzae,  340 
leprae,  364 
mallei  (of  glanders),  391 


Bacillus,  Holler's  grass,  367 
nitrifying,  528 
oedematis,  544 
of  bubonic  plague,  315 
pestis,  315 
pseudodiphtheriticum,  292,  336, 

415 

pyocyaneus,  309 
smegma,  3(54 
sporogenes,  557 

biology  of,  557 

morphology  of,  557 

pathogenesis  of,  558 
subtilis,  651,  665 
symptomatic  anthrax,  549 
tetani,  534 

tuberculosis,  179,  357 
typhosus,  426 
Bacteria,  52 
acid-proof,  365 
aerobic,  44 
anaerobic,  44 

methods  of  cultivating,  220 
behavior    of,    toward    staining 

reagents,  215 

capsule  surrounding,  50,  182 
chromogenic,  35 
classification  of,  52 
composition  of,  51 
conditions  necessary  to  growth, 

31-47 

constancy  in  morphology  of,  60 
definition  of,  31 
denitrifying,  36 
discovery  of,  17-19 
facultative,  33,  44 
fermentation  by,  215 

apparatus  for  testing,  216 

gases  resulting  from,  218 
flagellated  forms  of,  66 
identification  of,  193 
involution-forms  of,  60 
isolation  of,  in  pure  culture,  97 

on  slanting  media,  150 

plate  method,  145 

principles  of,  99 
metatrophic,  32,  33 
microscopic     examination     of, 

194,  204 

mode  of  multiplication,  57 
morphology  of,  49,  194 


INDEX. 


677 


Bacteria,  motility  of,  66 

nitrifying,  36,  528 

nutrition  of,  41 

parasitic,  32 

paratrophic,  32 

pathogenic,  35,  40,  559 
mode  of  action  of,  567 

photogenic,  35 

place  in  nature,  33 

points  to  be  observed  in  describ- 
ing, 194 

prototrophic,  32 

reaction  produced  by,  214 

reducing  power  of,  -30 

relation  to  man,  34 
to  oxygen,  44 
to  temperature,  45 

results  of  growth,  35 

role  in  nature,  33 

saprogenic,  36 

special  method  of  Metchnikoff, 
Roux,  and  Selembini  for 
cultivation,  265 

spore-formation  of,  63 
study  of,  207 

staining-reactions  of,  215 

structure  of,  49 

systematic  study  of,  193 

thermal  death-point  of,  67 

thermophilic,  46 

thiogenic,  36 

toxin  formation  by,  567 

very  minute,  method  of  ex- 
amination, 266 

zymogenic,  36 
Bacteriacese,  31,  54 
Bacterisemia,  200 
Bacterial  enzymes,  37 

proteins,  40,  565 

toxins,  564 

formation  of,  567 
point  of  action  of,  569 
Bacteriological  study  of  air,  640 
of  milk,  646 
of  soil,  645 
of  water,  616 

Bacteriology,       application       of 
methods  of,  267 

beginning  of,  17 
Barter iolysis,  568 
Bacterium,  54 


Bacterium  anthracis,  510 
diphtheria?,  399 
influenzas,  340 
leprse,  364 
mallei,  391 

biology  of,  393 
discovery  of,  391 
inoculation  of,  394 
staining  of,  395 
pneumonise,  33Q 
development  of,  333 
immune  serum,  336 
inoculation  of,  335 
morphology  of,  333 
staining  of,  334 
variation  of  virulence,  335 
where  found,  332 
pseudodiphtheriticum,  292,  336 
smegmatis,  364 
I  tuberculosis,  179,  357 
*Welchii,  556 
biology  of,  556 
morphology  of,  556 
pathogenesis  of,  557 
xerosis,  417 
Baumgarten,  371,  556 
Beggiatoa,  52 

Behring  and  Kitasato,  590 
Berkefeld  filter,  166 
Beyerinck,  35 
Billroth,  27 

and  Tiegel,  28 

Biochemic  characters,  194,  198 
Biologic  characters,  194,  195 
Birch-Hirschfeld,  26 
Blood,    Baumgarten's   views    on, 

612 
relations    to    bacteria    and    to 

toxins,  584 
Blood-serum  as  a  culture-medium 

(see  Media). 

germicidal  element  of,  587 
methods  of  obtaining,  126 
Nuttall's,  126 
Latapie's,  127 
Rivas's,  128 
preservation  of,  130 
Bolton's  potato  method,  118 
Bonnet,  24,  25  ^ 

Booker's     modification     of     Es- 
march's  method,  148 


678 


INDEX. 


Bouillon  (see  Media). 

Bresredka,  565 

Brieger  and  Cohn,  541,  566 

Brooding-over,  152 

Brownian  motion,  207 

Bubonic  plague,  315 

Buchner,  39,   40,  565,  573,  583, 

587 

Burdon-Sanderson,  29 
Burner,  Koch's  safety,  154 

nARBOLIC  acid  as  disinfectant, 

\J     94 

Chameleon  phenomenon  of  Ernst, 

312 

Characters  of  cultures,  194 
biochemic,  194, 198 
biological,  194,  195 
morphologic,  194 
Chauveau,  579,  580 
Chemical  composition  of  bacteria, 

51 

sterilization,  71,  87 
Chemotaxis,  47,  565 
Chevreul  and  Pasteur,  23 
Chlamydobacteriacese,  56 
Chlamydothrix,  56 
Chlorophyll,  31,  32 
Cholera  Asiatica,  diagnosis  of,  495 
method  of  Schottelius,  482 
microspira  of,  473,  474 
behavior  of,  in  butter,  493 
in  milk,  492 
in  soil,  491 
in  water,  490 
characteristics     of,    cultural, 

476 

morphologic,  474 
effects  of  drying,  494 
existence      outside     of     the 

body,  494 
experiments    upon     animals 

with,  485 
general  considerations  upon, 

488 

isolation  of,  482,  495 
location    in    the    body,  486, 

489 

morphology  of,  474 
persistence  of,  in  dead  body, 
491,  494 


Cholera,  microspira   of,  Pfeiffer's 

studies  upon,  483,  487 
poisons  produced  by,  483 
relation  to  gases,  481,  494 
to  other  bacteria,  482,  493 
to  putrefaction,  493 
to  sunlight,  491 
specific   reaction   of  immun- 
ized animals  to,  487 
toxin  of,  483 

Chromogenic  bacteria,  35 
Classen,  27 

Classification  of  bacteria,  52 
Cleaning  of  tubes,  etc.,  138 
Coagulating  enzymes,  37,  38 
Coccocese,  53 
Cohn,  25,  566 

Collodion  capsule  cultures,  211 
Colon  bacillus  (see  Bacillus  coli). 
Colonies,  appearance  of,  99,  101 
counting  of,  634 
formation  of,  99,  101 
study  of,  160,  195 
Colony  formation,  195 
Comma     bacillus      (see     Cholera 

Asiatica). 
Complements,  607 
multiplicity  of,  607 
origin  of,  607 
Cooling-stage,  145,  147 
\  Cornet,  355 

Corrosive  sublimate   as   disinfect- 
ant, 89 

Cover-slips,  cleaning  of,  168 
impression,  172 
microscopic     examination     of, 

204,  276 

preparation  of,  168 
steps  in  making,  169 
Crenothrix,  56 
Cultures,  agar,  195 
bouillon,  197 
collodion  capsule,  211 
gelatin,  196,  212 
hanging-block,  210 
hanging-drop,  205 
litmus  milk,  197 
potato,  196,  213 
pure,  162 
reactions  of,  214 
stab-  and  smear-,  162,  195 


INDEX. 


679 


Cultures,  test-tube,  162,  195 
Cygmeus,  432 


DAVAINE,  21 
Death-point,  thermal,  67 
Decolorizing  solutions,  178 
Decomposition,  33 
Defensive  proteids,  477,  589 
Denitrifying  bacteria,  36 
Diaphragm,  iris,  201 
Diastatic  enzymes,  37,  38 
Differential  diagnosis  with  aniline 

dyes,  214 

Differentiation  between  members 
of  the  bacterium 
diphtheria?  group, 
418 

Hiss's  media,  418 
Knapp's  method,  418 
Ljubinsky's      method, 

240 

Neisser's  method,  418 
Diphtheria  antitoxin,  422 
standardization  of,  424 
Behring's  method,  424 
Ehrlich's  method,  425 
bacterium  of,  399,401 

cultural  peculiarities  of,  403 
experiments  upon,  421 
location  in  tissues,  410 
method  of  obtaining,  399 
modification  in  virulence,  414 
morphology  of,  401 
pathogenesis  of,  408 
poison  produced  by,  412 
principles     of      immunizing 

against,  542 
staining,  408 
toxin  formation,  412 

potency  of,  566 

histological  changes  accompany- 
ing, 410 
Diplococci,  59 
Diplococcus  intracellularis  menin- 

gitidis,  330 
Disinfectants  and  antiseptics,  89, 

654 

experiments  with,  663 
general  considerations,  87 
methods  of  testing,  654 


Disinfectants,  methods  of  testing, 
precautions  to  be  observed, 
657 

mode  of  action,  89 
use  of  animals  as  test  objects 

for,  661 

in  the  laboratory,  94 
Disinfection,    general     considera- 
tions, 87 

influence  of  temperature  on,  91 
inorganic  salts  in,  92 
in  the  laboratory,  94 
investigations   of    Kronig    and 

Paul  on,  91 
modus  operand^  91 
reliable  agents  for  purposes  of, 

94 
selection  of  agents  to  be  used 

in,  88 

Dissociation,  electrolytic,  48,  91 
Dunham's  solution,  133 
Durham's  fermentation  tube,  219 

milk-whey,  132 
Dysentery,  bacilli  of,  463-472 
agglutination  of,  469 
cultural  peculiarities  of,  466- 

469 

discovery  of,  463 
immune  serum,  471 
morphology  of,  465 
pathogenesis,  467 
protective  inoculation,  470 
staining,  465 


"P  BERTH,  27 

_Cj     Ehrlich,  27,  574 

"  side-chain  "  theory  of  im- 
munity of,  600 
and  Morgenroth,  603 
Electrolytes,  48,  91 
Electrolytic  dissociation,  48,  91 
p]mboli  of  micrococci,  277 
Emmerich  and  Fowitzky,  599 

and  Low,  593 

and  Mattei,  592 
Endotoxins,  568 

liberation  of,  568 

point  of  action  of,  569 
Enzymes,  36,  37,  102 

bacterial,  37 


680 


INDEX. 


Enzymes,   bacterial,    coagulating. 

37 

diastatic,  37 
inverting,  37 
proteolytic,  37 
sugar-splitting,  37 
Ernst,  chameleon  phenomenon  of. 

312 

Erysipelas,  285 
Escherich,  432 
Esmarch's  counter,  638 
potato  method,  119 
tubes,  147 

Booker's  method   of   rolling, 

148 

made  of  agar-agar,  149 
Eubacteria,  52 
Examinations,  bacteriological,dur- 

ing  life,  263 
Exhaustion  hypothesis  of  Pasteur, 

581 

Exposure      and      contact-experi- 
ments, 269 
External    agencies,   influence   of, 

198 
Eye-piece,  201 

FACULTATIVE  bacteria,  33, 41 
Families,  bacterial,  52 
bacteriacese,  54 
chlamydobacteriaceae,  56 
coccaceae,  53 
spirillacese,  55 
Fehleisen,  27 

Fermentation,  33,  36,  39,  215 
gases  resulting  from,  218 
particular  forms  of,  36,  37 
Fermentation-tube,  216,  219 

method  of  using,  216 
Ferments,  36 
Fermi,  38 
Filling  tubes,  139 
Filter,  method  of  folding,  111 
Filtration  of  cultures,  233 
Fission  fungi,  49 
Flagella,  66 

methods  of  staining,  186 
Bunge's,  188 
Duckwall's,  189 
Loffler's,  186 
von  Ermengen's,  191 


Flagellated  organisms,  66 
Flasks,  preparation  of,  138 
Flexner,  388,  464 
Fluids,     examination    of,    during 

life,  263 
Fodor,  585,  588 
Foulerton,  289 
Fowl  tuberculosis,  372 
Frankland,  G.  and  P.  F.,  530 
Fungi,  fission,  49 
Funnel    for   tilling    aerobioseope, 
645 

for  filling  test-tubes,  140 

for  filtering  cultures,  660 

hot-water,  112 

GABBP7TFS  method,  181 
Gas-pressure  regulator,  158 
Gelatin,  cultures  in,  212 
liquefaction  of,  102 

characteristics  of,   164,212 
preparation  of  (see  Media), 
properties  of,  99 
Geppert,  90 
Germicide,  89 
Genus  bacillus,  55 
bacterium,  54 
chlamydothrix,  56 
crenothrix,  56 
micrococcus,  53 
microspira,  55 
phragmidiothrix,  57 
planococcus,  54 
pseudomonas,  55 
sarcina,  53 
sphserotilus,  57 
spirillum,  55 
spirochseta,  56 
spirosoma,  55 
streptococcus,  53 
Glanders,  389 
bacterium  of,  391 
cultivation  of,  393 
inoculation  with,  394 
morphology  of,  391 
staining  of,  in  tissues,  395 
diagnosis      of,      by       Strauss's 

method,  397 
by  use  of  mallein,  398 
manifestations  of,  389 
histology  of,  390 


INDEX. 


681 


Glanders,  susceptibility  of  animals 

to,  389 

synonyms,  389,  391 
Glass  plates,  145 
Gonococcus,  292 

appearance  in  pus,  293 
cultivation  of,  294 
Bumm's  method,  294 
Lipschiitz's  method,  299 
Wassermann's  method,  298 
Wertheim's  method,  294 
Wright's  method,  295 
distinguishing  features  of,  302 
morphology  of,  292,  300 
organisms  that  simulate  it,  301 
pathogenesis  of,  301 
vitality  of,  300 
Gonorrhoea,  pus  of,  293 
Gorini,  38 

Grass  bacillus  of  Moller,  367 
Green   pus   bacillus    (see  Pseudo- 
monas  aeruginosa). 

TTAFFKINE     vaccine    against 
II  plague,  325,  575 
Hanging-block  cultures,  210 
Hanging-drop,  205,  207 
Ilankin,  572,589 

and  Martin,  588 
Harvey,  25 
Henle,  22 

Hiss's  media,  136,  418,  441 
Hoffman,  23 
Hot- water  funnel,  112 
Hydrogen,  test  for  purity  of,  224 

sulphide,  test  for,  231 
Hypodermic  syringes  and  needles, 

243,  247 

Hypothesis,   exhaustion,  of    Pas- 
teur, 581 

retention,  of  Chauveau,  580 

IMMUNITY,  574 
J      acquired,  574 

blood  in,  585 
active,  575 

conclusions  concerning,  607 
earlier  studies  on  blood  rela- 
tive to,  585 

Ehrlich  and  Morgenroth,  (503 
Ehrlich's  theory  of,  COO 


Immunity,  "exhaustion"  hypoth- 

esis/581 
experiments  of  Klemperers  on, 

596 
hypothesis  of  Buchner,  59 1 

of  Emmerich  and  Low,  593 
mechanism  of,  579 
natural,  574 
nature  of  protective  bodies,  571, 

603 
observations   of     Behring    and 

Kitasato,  591 
passive,  575 

"  retention  "  hypothesis,  580 
theory  of  Metchnikoff,  582 
Impression     cover-slip      prepara- 
tions, 172 
Incubator,  152 

burner  for  heating,  154 
Indol,  method  of  detecting,  228 
production      of,     by    bacteria. 

227 
Infection,  559 

chemical  nature  of,  563 
conclusions  concerning,  607 
defense  of  body  against,  571 
modus  operandi,  569 
poisons  present  in,  567 
Influence    of    external    agencies, 

198 

Influenza,  bacterium  of,  340 
cultivation  of,  343 
dissemination  of,  344 
isolation  of,  from  tissues,  344 
morphology  of,  342 
occurrence  of,  in  tissues,  344 
staining  of,  342 
susceptibility  of  animals  to, 

345 

vitality  of,  344 
Inoculation  of  animals,  235 
apparatus  used  in,  237-241 
intralymphatic,  247 
intraocular,  250 
intraperitoneal    and    pleural, 

248 

intra vascular,  242 

subcutaneous,  235 

Inoculations,  agar  slant,  195 

stab,  196 
gelatin  stab,  196 


682 


INDEX. 


Inverting  enzymes,  37,  38 

Involution  forms  of  bacteria,  60 

Ions,  48,  91 

Iris  diaphragm,  201 

Isolation  of  bacteria  in  pure  cul- 
tures, 97 
Esmarch's    roll-tube 

method,  147 
Petri  plate  method,  145 
serial-tube  method,  150 

TOKDAN  and  Richards,  530 
V     Justinian  plague,  316 

T7ITASATO,  534,  540,  550 

JV     Klebs,  27,  29 

Klein,  557 

Klemperer,   F.  and  G.,  work  on 

pneumonia,  596 
Koch,  fundamental  researches,  29 

postulates  of,  357 

safety-burner,  154 

steam-sterilizer,  81 
Kronig  and  Paul,  91 

T  ACTOSE-LITMUS    agar-agar 
JU     or  gelatin  (see  Media). 
Latapie  apparatus,  127 
Leeuwenhoek,  17-19 
Lens  for  counting  colonies,  636 
Lepra  bacillus,  364 
staining  of,  370 
Leptothrix,  56 
Letzerich,  27 
Levelling-tripod,  145 
Liborius,  544 
Lime,  chloride  of,  96 

milk  of,  95 

Litmus  milk  (see  Media). 
Loffler's  alkaline  methylene-blue, 
175 

blood-serum  mixture,  123,  136 

method  of    isolation  of   bacte- 
rium diphtherise,  399 

stain  for  flagella,  186 
Loffler  and  Schiitz,  discovery  of 

bacterium  mallei,  391 
Lophotrichic  flagella,  66 
Lukomsky,  27 
Lumbar  puncture,  307 
Lysin,  279 


MADSEN,  542 
Malignant  oedema,   bacillus 

of,  544 
cultural  peculiarities  of, 

546 

morphology  of,  545 
pathogenesis  of,  547 
susceptibility  of  animals 

to,  548 
Mallein,  393 
Meat-extracts     in    culture-media, 

109 

Meat-infusion,  104 
Media,  culture-,  104 
agar-agar,  114 

clarification  of,  115 
filtration  of,  115 
glycerine,  116 
neutralization  of,  114 
solution  of,  115 
blood-serum,  120 

Councilman     and      Mallory 

method,  125 

mixture  of  Loffler,  123,  136 
Nu Mall's  method,  126 
original     method    of    Koch, 

120 
preservation  of,  130 

by  chloroform,  130 
sterilization     and     solidifica- 
tion of,  122 
bouillon,  104 

neutralization  of,  104 
gelatin,  109 

clarification  of,  112 
filtration  of,  110 
solution  of,  110 
sterilization  of,  113 
Hiss's    medium    of,    136,   418, 

441 
lactose-litmus     agar-agar     and 

gelatin,  135 

Lipschiitz's  medium,  299 
litmus-agar-agar,  131 
litmus-milk,  132 
litmus-whey,  132 
meat  infusion,  104 
milk,  131 
peptone     solution,     Dunham's, 

133 
potatoes,  117 


INDEX. 


683 


Media,  culture-,  potatoes,  Bolton's 

method,  118 
Esmarch's  method,  119 
mashed,  119 
original  method,  117 
Proskauer  and  Capaldi's,  444 
special,  131 

growth  in,  197 

Meningitis,    cerebrospinal,   causa- 
tive organism  of,  303 
lumbar  puncture  in,  307 
Metatrophic  bacteria,  32 
Metchnikoff,  582 

phagocytosis  theory  of,  582 
Roux    and    Selembini,  method 

of,  265 

Methods  of  isolating  bacteria,  97 
of  separating  bacteria,  142 
Esmarch's,  147 
Koch's  plate,  142 
Petri  dish,  145 
serial  tube,  156 
of  staining  bacteria,  167 
Gabbett's,  181 
glacial  acetic  acid,  182 
Gram's,  182 
spores,  183 
tubercle  bacteria,  179 
of  standardization  of  diphtheria 

antitoxin,  424 
Bern-ing's,  424 
Ehrlich's,  425 
Micrococci,  57 
emboli  of,  277 

mode  of  multiplication  of,  62 
Micrococcus,  53 
aureus,  271 
citreus,  280 

gonorrhoea  (see  Gonococcus). 
intracellularis,  303 
cultivation  of,  304 
diagnosis,    by    lumbar   punc- 
ture, 307  * 
morphology  of,  303 
powers  of  resistance,  307 
results  of  inoculations,  306 
lanceolatus,  330 
pyogenes,  280 
tetragenus,  337 
Microscope,  parts  of,  201 
Abbe  condenser,  203 


Microscope,    adjustment,    coarse, 

202 

fine,  203 

condenser,  Abbe,  203 
diaphragm,  iris,  201 
eye-piece,  201 
iris  diaphragm,  201 
nose-piece,  201 
objective,  201 
ocular,  201 
oil  immersion,  203 
reflector,  201 
stage,  201 

substage  condenser,  201 
Microspira,  55 
comma,  474 
Metchnikovi,  501 
biology  of,  501 
morphology  of,  501 
pathogenesis,  504 
Schuylkilliensis,  506 

biochemical     characters     of, 

508 

biology  of,  507 
morphology  of,  506 
pathogenesis  of,  508 
Milk  (see  Media), 
coagulation  of,  38,  39 
study  of,  646 
Mode  of  multiplication  of  bacteria, 

57 

Monotrichic  flagella,  66 
Morphologic  characters,  197 
Morphology  of  bacteria,  49,  197 
Moller's  grass  bacillus,  367 
Morgenroth,  603 
Motility  of  bacteria,  66 
Multiplication  of  bacteria,  mode 
of,  57 

N'AGELI,  41 
Nassiloff,  27 
Needham,  22 

Neisser,  gonococcus  of,  292 
Neisser's  stain,  418 
Neutralization    of   culture-media, 

104-109 
Nicolaier,  534 
Nitrification,  528 
Nitrifying  bacteria,  36,  528 
Nitrites,  tests  for,  231 


684 


INDEX. 


Nitro-rnonas  of  Winogradsky,  530 
c-ultural  pecularities  of,  531 
morphology  of,  531 
Nocard  and  Koux,  264 
Normal  serum,  289 

solution,  218 
Nutrition  of  bacteria,  41 
Nuttall,  126,  260,  320,  536,  586 
NuttalPs  bulb,  127 

OBJECTIVE,  201 
Ocular,  201 
Oertel,  27 
Ogata,  588 

Oil-immersion  system,  use  of,  204 
Organisms,  nitrifying,  528 
pathogenic,  35^  40,  1 99 
pyogenic,  271 
less  common,  280,  291 
Orth,  27 

Oven,  incubator,  152 
Oxygen,  relation  of  bacteria   to, 

44 
cultivation  of  bacteria  without, 

220 

Buchner's  method,  222 
FrankeP s  method,  223 
Hesse's  method,  221 
Koch's  method,  220 
Liborius's  method,  221 
Ozanam,  21 

PARASITE,  32 

L      Paratrophic  bacteria,  32 
Parietti's  solution,  627 
Pasteur,  21,  23,  29,  44,  544,  581 

exhaustion  hypothesis  of,  581 
Pathogenic  organisms,  35,  40,  199 

mode  of  action  of,  567 
Pepsin,  37 

Peptone,    Dunham's    solution   of, 
133 

test  of  purity  of,  134 
Peritonitis,  production  of,  274 
Peritrichic  flagella,  66 
Petersen,  279 
Petri  dishes,  146 
Pfeiffer,  340,  595,  602,  609 
Pfeiffer's  phenomenon,  594,  602, 

609 


Phenomenon  of  Pfeiffer,  594,  602, 

609 

of  Ernst,  312 
Phagocytosis  theory  of  Metchni- 

koff;  582 

Photogenic  bacteria,  35 
Phragmidiothrix,  57 
Pigments,  tests  with,  231 
Plague,  bubonic,  bacillus  of,  315 
cultivation  of,  318 
curative  serum,  323 
Haffkine  vaccine,  323 
immunity  from,  322 
mode  of  infection  with,  321 
morphology  of,  317 
occurrence  in  tissues,  321 
pathogenesis,  319 
vitality  of,  319,  320 
Planococcus,  53 
Planosarcina,  54 
Plates,    apparatus    employed    in 

making,  142 
Esmarch's  modification, 

147 

Booker's     modifica- 
tion, 148 

Koch's  fundamental  ob- 
servations, 97 
materials  used  in  making,  142 

Petri's  modification,  145 
principles     involved      in     the 

method,  97 

technique  in  making,  142 
Platinum  needles  and  loops,  143 
Plenciz,  20 

Pleuro-pneumonia  of  cattle,  265 
Pollander,  21 

Post-mortem  examination  of  ani- 
mals, 258 

cultures  from  tiasues  of,  260 
disinfection  of  implements 

after,  262 
disposal  of  remains  from, 

262 
incision  through    the  skin 

at,  258 
Nuttall's  spear  for  use  at, 

261 
opening   of  body   cavities, 

259 
position  during,  258 


INDEX. 


685 


Postmortem  examination  of  ani- 
mals, precautions  during, 
258 
preparation  of  cover-slips 

at,  262 
preservation   of    materials 

after,  262 

Postulates  of  Koch,  357 
Potato,  characteristics  of  cultures 

on,  213 

preparation  of,  for  culture  pur- 
poses (see  Media). 
Practical  disinfection,  94 
Precipitins,  577 

Preservation  of  blood-serum,  130 
Products  of  bacteria,  40 
Proskauer  and  Capaldi,  627 
Proteins,  bacterial,  40,  565 
Proteolytic  enzymes,  35,  37 
Prototrophic  bacteria,  32 
Prudden,  351 

Pseudodiphtheria  bacterium,  415 
Pseudomonas,  55 
aeruginosa,  309 

chameleon    phenomenon   of, 

312 

cultural  characters,  309 
enzymes  of,  313 
morphologic  characters,  309 
pathogenic  properties,  314 
protective  properties  of,  315 
Ptomains,  41,  565 
Pure  culture,  102 
Pus,   microscopic  appearance  of, 

271,  280 

Putrefaction,  33,  36,  46 
Pyaemia,  production  of,  274 
Pyogenic  organisms,  271,  280,  291 

QUARTER  evil  or  quarter  ill 
(.see  Symptomatic  anthrax ). 

p  ABINOVITCH,  36,367 

Lt    Reaction  of   media,  changes 

'in,  214 

Receptors,  605,  608 
Recklinghausen,  26,  27 
Reducing  power  of  bacteria,  230 
Reflector,  201 

Regulator,  gas-pressure,  158 
thermo-,  155 


Rennet,  38 

Retention  hypothesis,  580 

Rindfleisch,  26 

Rivas  apparatus,  128 

Roux  and  Yersin,  413,  566 


SAFETY  burner,  154 
Saprogenic  bacteria,  36 
Saprophyte,  32 

role  of,  in  nature,  33 
Sarcina,  53,  58 

mode  of  multiplication,  59,  62 
tetragena,  337 

cultural  peculiarities,  338 
morphology  of,  338 
susceptibility  of  animals,  340 
where  found,  337 
Scheme  of  study  of  bacteria,  194 
Schizomycetes,  31,  49,  52 
Schottelius's  method  of  examin- 
ing cholera  evacuations,  482 
Schroder  and  Dusch,  23 
Schrotter,  309 
Schulze,  23 
Schwann,  23 
Sections,  study  of,  276 
Separation  of  bacteria,  142 
Esmarch's  method,  147 
Koch's  plate  method,  142 
Petri  dish  method,  145 
serial  tube  method,  150 
Septicaemia,  327,  562 
Septica?mias,  560,  563 

hemorrhagic,  563 
Serum,  antidysenteriae,  471 
antiplague,  323 
an ti streptococcus,  287 
antitoxic,  diphtheria,  422 

tetanus,  542 
blood-,    methods  of    obtaining, 

126 

of  preserving,  130 
Serum-water  media  of  Hiss,  136 
Sewage  streptococcus,  639 
Shiga's  bacillus  (.see  Dysentery). 
"Side-chain"   theory  of  Ehrlich, 

600 
Skin  disinfection,  experiments  in, 

666 
Smear-cultures,  162 


686 


INDEX. 


Smegma  bacillus,  staining   pecu- 
liarities of,  364 
Smith,  Theobald,  374 
Soil,    bacteriological    analysis   of, 
645 

nitrifying  bacteria  in,  528 

organisms  present  in,  528 

phenomena  in  operation  in,  528 
Spallanzani,  22,  23,  24,  29 
Special  media,  131 
growth  in,  197 
Sphserotilus,  57 
Spirilla,  58 
Spirillaceae,  55 
Spirillum,  55 

of  Asiatic  cholera  (we  Cholera). 

of  Metchnikovi  (see  Microspira 
Metchnikovi). 

Schuylkilliensis  (see  Microspira 

Schuylkilliensis). 
Spirochseta,  56 
Spirosoma,  55 
Spores,  formation  of,  61,  63 
method  of  studying,  207 

mode  of  development  of,  63,  66 

recognition  of,  61,  64 

staining  of,  183 

Sputum,   inoculations   with,  327, 
329 

microscopic     examination      of, 
327 

pathogenic  properties  of,  329 

septicaemias,  327,  330 

tuberculous,  315,  327 
Stab-cultures,  162 
Staining,   methods  and  solutions 
used  in,  167 

acetic  acid,  182 

Bunge's,  188 

Duckwall's,  189 

Gabbett's,  181 

general  remarks  on,  177 

Gram's,  182 

Koch.Ehrlich's,  175 

Loffler's  blue,  175 
flagella,  185 

Holler's,  185 

of  spores,  183 

of  tubercle  bacteria,  174 

ordinary  solutions  used  in,  173 
bottles  for  holding,  174 


Staining,  van  Ermengem's,  191 

Ziehl-Nielsen's,  176 
Staphylococci,  58 
I  Stnphylococcusepidermidis  albus, 

281 

pyogenes  albus,  280 
aureus,  271 

cultural  characters   of,  272 

pathogenesis,  274 

toxin  of,  279 

where  to  be  expected,  273, 

285 

citreus,  280 
Staphylotoxin.  278 
Sterilization,  chemical,  76,  87 
by  heat,  69,  71 

principles  involved,  71 
by  hot  air  or  dry  heat,  85 
apparatus  used  in,  86 
by  steam,  72 

apparatus  used  in,  83,  84 
discontinued,  73,  75 
fractional,  75,  78 
under  pressure,  79,  83 
experiments  upon,  649 
intermittent,  73,  75 

at  low  temperature,  78 
methods  employed  in,  72 
principles  involved  in,  71-84 
use  of  the  term,  69 
Sternberg,  332 
Strauss's  method  for  diagnosis  of 

glanders,  397 
Streptococci,  58 

mode  of  multiplication,  59 
Streptococcus,  53 
pyogenes,  281,  285 
biology  of,  281-285 
curative  serum,  287 
effects  of,  in  inoculation,  285 
longus  and  brevis,  287 
morphology  of,  281 
where  found,  281,  285 
of  sewage,  639 

Streptothrices,  pathogenic,  376 
Streptothrix,  56 
Subtilis  bacillus,  651,  660 
Sugar-splitting  enzymes,  37,  39 
Suppuration,  271 

bacteria  common  to,    271,  280, 
291 


INDEX. 


687 


Suppuration,     general      remarks 

upon,  291 

less  common  causes  of,  291 
microscopic  appearance  of  pus, 

270,  280,  292 
Symbiosis,  36,  46,  528 
Symptomatic  anthrax,  bacillus  of, 

549 

biology  of,  551 
differentiation  from   bacil- 

cillus  oedematis,  555 
morphology  of,  550 
pathogenesis  of,  554 
susceptibility  of  animals  to, 
555 


TECHNIQUE,  novel,  226 
Test-tubes,  cleaner  for,  138 
cleaning  of,  138 
filling  with  media,  139 
apparatus  for,  140 
plugging  with  cotton,  129 
position  after  tilling,  141 
sterilization  of,  141 
Tests  for  ammonia,  232 
for  hydrogen  sulphide,  231 
for  indol,  228 
for  nitrites,  231 
for  toxins,  233 
with  pigments,  231 
Tetanolysin,  542 
Tetanospasmin,  542 
Tetanus  antitoxin,  542 
bacillus  of,  534 
biology  of,  536 
effects  of,  on  animals,  539 
method  of  obtaining,  534 
morphology  of.  536 
poison  produced  by,  540 
toxin,  composition  of,  542 

potency  of,  541,  566 
Tetrads,  58 
Theory,  alexin,  583 
phagocytosis,  582 
"side-chain,"  600 
Thermal  death-point  of   bacteria, 

67 

Thermophilic  bacteria,  46,  77 
Thermo-regulator,  155 
Thermostat  (see  Incubator). 


Thiogenic  bacteria,  36 
Tissues,    cultures    from,    at     au- 
topsies, 260 

examination  during  life,  263 
NuttalPs  spear  for  making, 

260 

Toxemia,  199,  562 
Toxin   molecule,    Ehrlich's    con- 
ception of,  566 
Toxins,  41,  564 
formation  of,  567 
point  of  action,  567 
Toxoids,  541,  566 
Toxones,  541,  566 
Traube  and  Gseheidlen,  584 
Treviranus,  22,  29 
Tripod  for  levelling  plates,  144 
Trypsin,  37 
Tube,  Esmarch,  147 
Tuberculin,  375 
Tuberculosis,  346-376 
avian,  372 
bacterium  of,  357 

appearance  in  cultures,  361 
cultivation  from  tissues,  358 
methods     of    staining,    179, 

362 

Gabbett's,  181 
Koch-Ehrlich's,  175, 179 
Ziehl-Nielsen's,  176,  180 
microscopic    appearance    of, 

330 
organisms   that  simulate    it, 

363 
differential  diagnosis  of, 

370 
staining  peculiarities  of,  179, 

329,  362 
toxin  of,  565 
varieties  of,  374 
cavity-formation  in,  350 
condition  simulating,  375 
diffuse  caseation  of,  375 
encapsulation      of      tubercular 

foci,  352 

giant  cells  in.  349 
location  of  bacteria  in,  355 
manifestations  in  experimental, 

347 

miliary  tubercle,  structure   of, 
348 


688 


INDEX. 


Tuberculosis,  modes  of  infection, 

353 

primary  infection,  352 
pseudo-,  375 
sputum  in,  315 

inoculation  of  animals  with, 

329 
microscopic    appearance    of, 

330 

staining  of,  179,  329 
susceptibility    of    animals     to, 

375 

vaccination  against,  376 
Tubes,  Esmarch  roll,  147 
fermentation,  217,  219 
filling  of,  139 
preparation  of,  138 
serial,  150 
Tyndall,  24 
Typhoid  fever,  bacillus  of,  426 

constant      properties       of, 

435 

cultivation  of,  427 
differentation  from  bacillus 

coli,  448,  457 
Drigalski   and  Conradi's 

method,  445 
Fischer's  method,  451 
Hiss's  method,  441 
Hoffmann   and   Picker's 

method,  449 
Parietti's  method,  627 
Proskauer  and  Capaldi's 

method,  444 
difficulty     in     identifving, 

435 

experiments  with,  452 
inoculations  with,  431 
isolation  from  cadavers, 

452 

location  of,  in  tissues,  430 
morphology  of,  426 
reaction  of,  with    typhoid 
serum,  436 

source  from  which  to   ob- 
tain, 452 

vaccination  against,  576 
water    as     a     carrier    of, 

441,  616 

Widal's  reaction  with,  431, 
436,  578 


TTACCINATION    against     dis- 

V     eases,  575 
Vaccines,  575 

plague,  Haffkine  method,  325 
typhoid  fever,  Wright  method, 

576 

Vaughan,  589 
Vegetable  alkaloids,  41 
Vibrio  Metchnikovi,  501 

characteristics     of,    cultural, 

501 

morphological,  501 
pathogenesis,  504 
Schuylkilliensis,  506 
biochemistry  of,  508 
biology  of,  507 
morphology  of,  506 
pathogenesis  of,  508 
Vibrion  septique,  549 


WALDEYER,   26 
Wasserman,    studies    on     te- 
tanus toxin,  569 

Water,  general  observations  upon 
bacteriological       study       of, 

616 

qualitative   bacteriological    an- 
alysis of,  622 
precautions  in  obtaining 

sample,  623 
preliminary     steps      in, 

623 
quantitative         bacteriological 

analysis  of,  629 
collection      of     sample, 

630 

counting      of      colonies, 
634 

apparatus  for,   635, 

639 

reaction  of  media,  633 
selection   of  proper  me- 
dium for,  632 

relation  of,  to  epidemics,  616 
sewage  streptococcus,  639 
typhoid  bacilli  in,  616,  619 
value  of  bacteriological   exam- 
ination of,  618 

of  chemical  examination  of, 
618 


INDEX. 


689 


Weichselbaum,  303,  330  Wright,  295,  382,  576 

Weigert,  30,  600  ,  Wurtz's  agar-agar  and  gelatin,  135 

doctrine     of     cell-equilibrium, 

600  !  YEROSIS  bacterium,  417 

Welch,  281,  291,  331,  556  A 

Widal's   reaction,   431,  437,  578, 

609  VERSIN>  322>  566 

Wilde,  27  I 

Winogradsky,  nitro-monas  of,  530 
Wolfl  hiigePs  counting-apparatus,     yOOGLCEA  of  bacteria,  50 

635  ZJ    Zymase,  39 

Wound-infection,  26-29  ;  Zymogenic  bacteria,  36 

H 


THIS  BOOK  IS  DUE  ON  THE  LAST  DATE 
STAMPED  BELOW 


AN  INITIAL 

WILL   BF" 


BIOLOGY 
yBRARY 

UNIVERSITY  OF  CALIFORNIA  LIBRARY 


